Category Archives: Abstracts

Rod Bipolar Cell Networks in Early Retinal Remodeling

Rebecca Pfeiffer, a post-doc in the laboratory presented her work on “Rod Bipolar Cell Networks in Early Retinal Remodeling” as a platform presentation at the ISER 2018 meeting in Belfast, Northern Ireland.

Authors: Rebecca Pfeiffer, James R. Anderson, Daniel P. Emrich, Jeebika Dahal, Crystal L Sigulinsky, Hope AB Morrison, Jia-Hui Yang, Carl B. Watt, Kevin D. Rapp, Jessica C Garcia, Mineo Kondo, Hiroko Terasaki, Robert E. Marc, and Bryan W. Jones.

Abstract: Retinal remodeling is a form of negative plasticity that occurs as a consequence of retinal degenerative diseases. Part of retinal remodeling involves anomalous sprouting of processes, termed neurites. The synaptic structures and partners of the neurites are not yet defined, leading to uncertainty about the consistency of network motifs between healthy and degenerate retina. Our goal is to map out the identities and network relationships of bipolar cell networks using a connectomics strategy. Retinal connectomes or ultrastructural maps of neuronal connectivity have substantially contributed to our understanding of retinal network topology, providing ground truth against which pathological network topologies can be evaluated. We have generated the first pathoconnectome (RPC1), or connectome of pathological tissues, of early retinal remodeling at 2nm/pixel, and are currently investigating the impact of remodeling on network architecture.
The tissue for RPC1 was obtained from a 10mo transgenic P347L rabbit model of autosomal dominant retinitis pigmentosa. Tissue was fixed in mixed aldehydes, osmicated, dehydrated, embedded in epon resin, and sectioned at 70nm. Serial sections were placed on grids, stained, and imaged using a JEOL JEM-1400 TEM using SerialEM software. Every 30th section was reserved for computational molecular phenotyping (CMP), and probed for small molecules: glutamate, glutamine, glycine, GABA, taurine, glutathione; or TEM compatible proteins GFAP and GS. The pathoconnectome volume is explored and annotated using the Viking software suite.
RPC1 was selected as an example of early retinal remodeling, demonstrating Muller cell hypertrophy, metabolic dysregulation, and degeneration of rod outer segments, indicating phase 1 remodeling and neuronal sprouting. We have observed the presence of both cone pedicles and rod spherules within the OPL to be synaptically active with neurites from some rod bipolar cells forming functional synapses with both rod spherules and cone pedicles. These rod bipolar cells also exhibit structurally altered ribbon synapses. We are currently evaluating network motifs and comparing them to networks established from our previous connectome, RC1, generated from a healthy rabbit.
These findings allow us to evaluate and analyze the impact of retinal remodeling on retinal networks which may have important implications for therapeutic interventions being developed which rely on inner retina network integrity.

Pathoconnectome Analysis of Müller Cells in Early Retinal Remodeling

Rebecca Pfeiffer, a post-doc in the laboratory presented her work on “Pathoconnectome Analysis of Müller Cells in Early Retinal Remodeling” as a platform presentation at the RD2018 meeting in Killarney, Ireland.

Authors: Rebecca Pfeiffer, James R. Anderson, Daniel P. Emrich, Jeebika Dahal, Crystal L Sigulinsky, Hope AB Morrison, Jia-Hui Yang, Carl B. Watt, Kevin D. Rapp, Mineo Kondo, Hiroko Terasaki, Jessica C Garcia, Robert E. Marc, and Bryan W. Jones.

Purpose: Glia play important roles in neural system function. These roles include, but are not limited to: amino acid recycling, ion homeostasis, glucose transport, and removal of waste. During retinal degeneration, Muller cells, the primary macroglia of the retina, are one of the first cells to show metabolic and morphological alterations in response to retinal stress. The metabolic alterations observed in Muller cells appear to manifest in regions of photoreceptor degeneration; however, the precise mechanisms that govern these alterations in response to neuronal stress, synapse maintenance, or glia-glia interactions is currently unknown.  This project aims to reconstruct Muller cells from a pathoconnectome of early retinal remodeling at 2nm/pixel with ultrastructural metabolic data to determine the relationship of structural and metabolic phenotypes between neighboring neurons and glia.

Methods:  Retinal pathoconnectome 1 (RPC1) is the first connectome to be assembled from pathologic neural tissue (a pathoconnectome). The tissue selected for RPC1 was collected post mortem from a 10 month transgenic P347L rabbit model of autosomal dominant retinitis pigmentosa, fixed in 1% formaldehyde, 2.5% glutaraldehyde, 3% sucrose, and 1mM MgSO4 in cacodylate buffer (pH 7.4). The tissue was subsequently osmicated, dehydrated, resin embedded, and sectioned at 70nm. Sections were placed on formvar grids, stained, and imaged at 2nm/pixel on a JEOL JEM-1400 TEM using SerialEM software. 1 section was reserved from every 30 sections for CMP, where it was placed on a slide and probed for small molecules: glutamate, glutamine, glycine, GABA, taurine, glutathione; or TEM compatible proteins GFAP and GS. The pathoconnectome volume was evaluated and annotated using the Viking software suite.

Results: RPC1 demonstrates hallmarks of early retinal degeneration and remodeling, including the glial phenotypes of hypertrophy and metabolic variation between neighboring Muller cells. Early evaluation of these glia demonstrates variations in osmication in Muller cells as well as apparent encroachment of glial end-feet on one another.  We are currently in the process of reconstructing multiple Muller cells within RPC1 and their neighboring neurons.  Once complete, we will assess the relationship between Muller cell phenotype and the phenotypes of contacted neuronal and glial neighbors.

Conclusions: How neural-glial relationships are affected by retinal remodeling may help us understand why remodeling and neurodegeneration follow photoreceptor degeneration. In addition, determining these relationships during remodeling will be crucial to developing therapeutics with long-term success. RPC1 provides a framework to analyze these relationships in early retinal remodeling through ultrastructural reconstructions of all neurons and glia in an intact retina. These reconstructions, informed by quantitative metabolite labeling, will allow us to evaluate these neural-glial interactions more comprehensively than other techniques have previously allowed.

Coupling Architecture Of The Aii/ON Cone Bipolar Cell Network In The Degenerate Retina

Crystal Sigulinsky, a post-doc in the lab, presented her work on “coupling architecture of the
Aii/ON cone bipolar cell network in the degenerate retina” at the RD2018 meeting in Killarney, Ireland today.  Authors are: Crystal L Sigulinsky, Rebecca L Pfeiffer, James R Anderson, Jeebika Dahal, Hope Morrison, Daniel P. Emrich, Jessica C Garcia, Jia-Hui Yang, Carl B. Watt, Kevin D. Rapp, Mineo Kondo, Hiroko Terasaki, Robert E. Marc, and Bryan W. Jones.

Purpose: Retinal network hyperactivity within degenerative retinal networks is a component of the disease process with implications for therapeutic interventions for blinding diseases that depend upon the surviving retinal network. Connexin36-containing gap junctions centered on the Aii amacrine cell network appear to mediate the aberrant signaling observed in mouse models of retinal degeneration. However, it remains unclear whether this hyperactivity reflects changes in the underlying circuitry or dysfunction/dysregulation of the normative circuitry. Mapping retinal circuitry in the ultrastructural rabbit Retinal Connectome, RC1, has revealed Aii network topologies explicitly involving gap junctions. In addition to canonical Aii-to-Aii and Aii-to-ON cone bipolar cell (CBC) coupling, we describe pervasive in- and cross-class coupling motifs among ON CBCs that extend and dramatically expand the coupled Aii network topologies. Since virtually every gap junction in the inner plexiform layer contains Connexin36, these circuits likely participate in the aberrant signaling of degenerate retinas. This study examines these Aii and ON CBC coupling motifs in Retinal PathoConnectome 1 (RPC1), an ultrastructural pathoconnectome of a rabbit model of retinitis pigmentosa.

Approach: RPC1 is a 2nm/pixel resolution volume of retina from a 10 month old, transgenic P347L rabbit model of autosomal dominant retinitis pigmentosa in early phase 1 retinal remodeling, a time point where cone and rod photoreceptors are still present, albeit going through cell stress. RPC1 spans the vitreous to basal outer nuclear layer and was built by automated transmission electron microscopy and computational assembly. ON CBCs, Aii amacrine cells, and their coupling partners were annotated using the Viking application and explored with 3D rendering and graph visualization of connectivity. Gap junctions were validated by 0.25 nm resolution recapture with goniometric tilt when necessary. Motifs were compared to those discovered in RC1. RC1 is a 2 nm resolution, 0.25 mm diameter volume of a light-adapted adult female Dutch Belted rabbit retina spanning the ganglion cell through inner nuclear layers.

Conclusions: RPC1 shows degeneration of rod outer segments, Müller cell hypertrophy and neuronal sprouting, characteristic of early stage retinal degeneration and phase 1 remodeling, when retinal hyperactivity and its reliance on gap junctional coupling has likely already initiated and human patients would still have some vision. All major coupling motifs (Aii-to-Aii, Aii-to-ON CBC, and ON CBC-to-ON CBC) were observed. Preliminary examinations indicate that several ON CBC classes retained their class-specific coupling profiles, accepting and rejecting specific combinations of Aii and ON CBC class partnerships. However, recent findings reveal aberrant partnerships in the coupled network, including both loss of prominent motifs and acquisition of novel ones. Thus, clear aberrant morphological and synaptic changes have been identified in RPC1, including changes in the coupling specificity and gap junction distributions of both Aii amacrine cells and ON CBCs (Figure 6). This suggests that the Aii/ON CBC circuit topology is already altered during early phase 1 remodeling, with substantial implications for therapeutic interventions in human subjects. The full coupling network is actively being examined and progress has begun on RPC2, a second pathoconnectome for examining later, phase 2 remodeling in this same model.

An almost full size poster available here in pdf format.

Structure-Function Correlation Across The Central Visual Field Using Pointwise Comparisons and Ganglion Cell Isocontours Derived From Pattern Recognition

We presented an abstract for 23rd International visual field and imaging symposium in Kanazawa, Japan on May 12th titled, Structure-function correlation across the central visual field using pointwise comparisons and ganglion cell isocontours derived from pattern recognition.  Authors are:

Kalloniatis M1,2, Tong J1,2, Yoshioka N1,2, Khuu SK2, Phu J1,2, Choi A1,2, Zangerl B1, Nivison-Smith L1,2, Bui BV4, Marc RE3, Jones BW3

  1. Centre for Eye Health, University of New South Wales (UNSW), Kensington, NSW, Australia.
  2. School of Optometry and Vision Science, UNSW, Sydney, NSW, Australia.
  3. Moran Eye Center, Univ of Utah, Salt Lake City, UT, United States of America.
  4. Department of Optometry and Vision Science, Univ of Melbourne, Parkville, Victoria, Australia

Purpose: To establish the correlation between visual field sensitivity and ganglion cell density within the central 20 degrees.  We hypothesized that the use of a test stimulus within complete spatial summation (Goldmann II, GII) would display improved correlation compared to the standard GIII test stimulus.

Methods:One eye of 40 normal subjectswas included in this study. The Humphrey Field Analyser (HFA) was used in full-threshold mode for the 10-2 test grid and 12 points from the 30-2 grid that matched the outer Spectralis grid. Spectralis OCT posterior pole scans for each subject was extracted and the average ganglion cell layer (GCL) thickness values were obtained for each of the 64 grid location within the measurement area ~6880µmx6880µm.  HFA sensitivity in dB was plotted against GC density/mm3(calculated from GCL thickness and GC density from histological data, also converted into dB). Both visual field and OCT data were converted to a 50 year-old equivalent for analysis. The Drasdo et al VR 2007 correction was applied to visual field data to allow comparison of structure and function (Fig. 1). Linear regression analysis was conducted at each test location using individual data or grouped data derived using the 5, 6, 7 and 8 GC iso-density theme classes of Yoshioka et al IOVS 2017 (Fig. 1). A non-parametric bootstrap was used to determine the 99% distribution limits of the slope and correlation parameters.

Results: Table 1 shows the structure-function correlation slope parameters and coefficients of determination (R2) for point-wise and theme class-based comparisons, using GII and GIII. The use of 5 or 6 theme classes resulted in a slope close to unity and high R2values for GII. Table 2 shows the 99% distribution of the slope parameters and R2values for point wise comparisons and those using 5 theme classes again demonstrating superior correlations for GII (both slope and R2 significantly different p<0.01 compared to pointwise analysis). Correcting the data for test size difference (6dB) did not result in data superposition confirming that GIII test size is not within complete spatial summation within the central 20 degrees.

Conclusions:Using a test stimulus within complete spatial summation (GII) and grouping sensitivities according to GC density test grids derived using pattern recognition (7 or fewer GC theme classes), revealed correlations close to unity with coefficients of determination (R2) >0.90. The high correlations achieved when using theme classes even when using individual datasets, suggests that an approach would provide a useful method to predict alterations of visual field sensitivity from OCT data.

Commercial Relationships Disclosure:MK and SKK commercial Relationship(s):2014/094035 A1 (USA) and 13865419.9 (EU):Code P (Patent): REM, JT, BZ, L N-S, BJ, RF: none

Grant support:  NHMRC 1033224;Guide Dogs NSW/ACT; NIH EY02576, EY015128, EY014800, an Unrestricted Grant to the Moran Eye Center from Research to Prevent Blindness.

A Pathoconnectome of Early Retinal Remodeling

This abstract was presented today, Monday, April 30th at the 2018 Association for Research in Vision and Opthalmology (ARVO) meetings in Honolulu, Hawaii by Rebecca Pfeiffer, Robert E. Marc, James R. Anderson, Daniel P. Emrich, Carl B. Watt, Jia-Hui Yang, Kevin D. Rapp, Jeebika Dahal, Mineo Kondo, Hiroko Terasaki, and Bryan W. Jones.

Retinal remodeling is a consequence of retinal degenerative disease, during which neurons sprout new neurites whose synaptic structures and partners are not yet defined. Simultaneously during remodeling, Müller cells (MCs) undergo structural and metabolic changes, whose impact on surrounding neurons is an active area of research. Retinal connectomes have elucidated and validated fundamental networks. These data provide further classification of neuronal types and subtypes and a precise framework for modeling of retinal function, based on ground truth networks. The creation of the first pathoconnectome (RPC1), a connectome from pathological retinal tissue, provides the opportunity to determine connectivites between neurons, while simultaneously evaluating glial remodeling. Computational Molecular Phenotyping (CMP) embedded within the ultrastructure provides metabolic factors of pathologies.

RPC1 was collected post-mortem from a 10mo TgP347L rabbit model of adRP, fixed in 1% FA, 2.5% GA, 3% sucrose, and 1mM MgSO4 in cacodylate buffer (pH 7.4). The tissue was osmicated, dehydrated, resin embedded, and sectioned at 70nm. Sections were placed on formvar grids, stained, and imaged on a JEOL JEM-1400 TEM using SerialEM. 1 section was reserved from every 30 section for CMP, where it was probed for small molecules: glutamate, glutamine, glycine, GABA, taurine, glutathione; or proteins GFAP and GS. RPC1 was evaluated using the Viking software suite.

RPC1 was chosen based on early features of retinal degeneration/remodeling: degeneration of rod OS, MC hypertrophy, and neuronal sprouting. RPC1 consists of 948 serial sections spanning the ONL to the vitreous, with a diameter of 90µm. We find dendrites extending from rod bipolar cells to cone pedicles, originally described in light microscopy, and active synaptic contacts. We also see alterations of synaptic structure in the IPL, and MC morphological changes affecting surface to volume and neuron/glial relationships. Network motifs are being actively investigated.

We observe many features of remodeling previously described using light microscopy, and confirm active synaptic contact. We also find synaptic structural features, not previously described. In addition, early evaluation of MC morphology demonstrates marked changes in MC shape and associations with nearby neurons and glia, which, combined with CMP, will be instrumental in understanding how MCs affect retinal remodeling.

Impact of Glaucoma On Retinal Ganglion Cell Subtypes: A Single-Cell RNA-seq Analysis of the DBA/2J Mouse

This abstract was presented today, May 1st at the 2018 Association for Research in Vision and Opthalmology (ARVO) meetings in Honolulu, Hawaii by Siamak Yousefi, Hao Chen, Jesse Ingels, Sumana R. Chintalapudi, Megan Mulligan, Bryan W. Jones, Vanessa Marie Morales-Tirado, Pete Williams, Simon W. John, Felix Struebing, Eldon E. Geisert, Monica Jablonski, Lu Lu, Robert Williams

We are developing methods to define molecular signatures of cellular stress during early stages of glaucoma for major subtypes of retinal ganglion cells (RGCs). Our first aim is to develop reliable mRNA biomarkers for RGC subtypes in the DBA/2J (D2) mouse model prior to disease onset. Our second objective is to quantify cellular stress in RGC subtypes at early stages of disease using known sets of stress-responsive transcripts (e.g. Struebing et al, 2016 PMID:27733864; Williams et al. 2017, PMID:28209901; Lu et al, ARVO 2018).

Whole retinas from D2 or D2.Cg-Tg(Thy1-CFP)23Jrs/SjJ at 130 to 150 days-of-age were dissociated gently and size selected (>10 µm). RGCs were enriched using THY1 antibody-coated beads. Fluidigm HT microfluidics plates were used to isolate and generate scRNA-seq libraries of full length polyA-positive mRNAs using SMART-Seq v4. Libraries were sequenced using HiSeq3000, PE151. Following alignment using STAR, expression was normalized to log2(FPKM+1) across ~25,000 unique transcript models. Cells with fewer than 1000 detected genes and genes expressed in fewer than 1% of RGCs were excluded. Sets of genes with high variance and/or high expression were used for principal component analysis (PCA). Twenty PCs were used for graph-based unsupervised clustering and visualized using t-distributed stochastic neighbor embedding (tSNE). Gene specificity was computed for all transcripts across all clusters. The top transcripts per cluster with expression >1 in 1% or more of cells, were used to diagnose cellular identify of clusters. The top 30 genes per cluster were searched in PubMed against a panel of cell and tissue specific terms using Chilibot.

The scRNA-seq protocol generates 150,000 – 200,000 uniquely mapped mRNA reads/cell and ~5000 genes/cells. We currently have 1600 cells, of which over half are RGCs. Around 75% of cells are positive for two or more of the following RGC markers: Thy1, Rbpms, Rbpms2, Jam2, G3bp1, and Ywhaz. This set of cells and different subsets of genes are now being used for RGC clustering. We have identified at least 17 clusters in initial datasets using these protocols and are now linking clusters to major classes of RGCs.

Molecular signatures of cellular stress and RGC subtypes in early stage of glaucoma should now be identifiable using unsupervised learning techniques.

Circuit Remodeling In Retinal Degeneration

This abstract was presented yesterday, April 29th at the 2018 Association for Research in Vision and Opthalmology (ARVO) meetings in Honolulu, Hawaii by Bryan W. Jones.


The retina is a complex, heterocellular tissue with most/all retinal cell classes becoming impacted or altered in retinitis pigmentosa (RP) and age-related macular degeneration (AMD) in a process called retinal remodeling. Defining disease and the stage-specific cytoarchitectural and metabolic responses in RP and AMD is critical for highlighting targets for intervention. We now know that negative plasticity and neural retinal remodeling occurs regardless of retinal insult in models of retinal degeneration as well as in human RP and in human AMD, revealing that no retinal disease fails to trigger remodeling and reprogramming.

Evidence in the literature over the past decade has improved our understanding into mechanisms of initial retinal degeneration and informed our understanding of the subsequent remodeling events in the neural retina that occur post-photoreceptor degeneration. Remodeling associated with retinal degeneration is intimately linked with insults that cause photoreceptor stress and eventually photoreceptor cell death. These phenomena result in reprogramming of cell types in retina followed by progressive neural degeneration akin to CNS neural degenerations involving both neuronal and glial classes. No cell class in the retina is spared from the effects of remodeling. The earliest cell classes involved in remodeling are horizontal, bipolar and Müller cells and the Müller glia are the last cell class left in the remodeling retina.

Our efforts are now focused on elucidating the precise wiring changes in retina, through the creation of pathological connectomes, or “patho-connectomes” to study precisely what the circuit topologies are, compared to normal topologies derived from Retinal Connectome 1 (RC1).  Also, because temporal windows are critical to understanding when interventions may be possible, we are exploring when circuit topology revisions occur to understand their impact on information flow in the retina and their impact on rescues of vision loss.  Precise circuit topologies in early retinal degenerative events is our first area of exploration with ultrastructural reconstructions of outer retinal neurons, bipolar cells and horizontal cells.  Müller glia are also of intense interest as we are tracking the earliest metabolic and morphological changes in glia in response to retinal degenerations.

Synaptic Inputs To A Gamma Ganglion Cell In Rabbit Retina

This abstract was presented today, May 8th at the 2017 Association for Research in Vision and Opthalmology (ARVO) meetings in Baltimore, Maryland by Andrea Bordt, Diego Perez, Robert E. Marc, James R. Anderson, Carl B. Watt, Bryan W. Jones, Crystal Sigulinsky, James S. Lauritzen, Danny Emrich, Noah Nelson, Luke Tseng, Weiley Liu, and David W. Marshak. Full resolution version here.

Purpose: There are at least 30 distinct types of mammalian retinal ganglion cells, each sensitive to different features of the visual environment, and these can be grouped according to their morphology. One such group, the gamma cells, was identified more than 40 years ago, but their synaptic inputs have never been described. That was the goal of this study.

Methods: The synaptic inputs to a subtype of gamma cell with dendrites ramifying in the outer sublamina of the inner plexiform layer (IPL) of the rabbit retina were identified in a retinal connectome developed using automated transmission electron microscopy.

Results: The gamma cell was always postsynaptic in the IPL, confirming its identity as a ganglion cell. The local synaptic input should produce relatively weak OFF reposnses to stimuli confined to the center of the gamma cell’s receptive field. It typically received only one synapse per bipolar cell from at least 4 types of OFF bipolar cells. Because bipolar cells vary in their response kinetics and contrast sensitivity. each type would provide a small, asynchronous excitatory input. The amacrine cells at the dyad synapses provided only a small amount presynaptic inhibition; reciprocal synapses were observed in only 3 of the 18 ribbon synapses. There was no glycinergic crossover inhibition, another local interaction that would enhance light responses. Local postsynaptic inhibition was somewhat more common; in 6 instances, the bipolar cells presynaptic to the gamma cell or their electrically coupled neighbors also provided input to an amacrine cell that inhibited the gamma cell. The other amacrine cell inputs to the gamma cell should have a much greater impact on the light responses because they are far more numerous. These are from axons and long dendrites of GABAergic amacrine cells, and they provide 60% of all the input. This finding suggests that many types of stimuli in the receptive field surround or outside of the classical receptive field would provide potent inhibition to the gamma cell.

Conclusions: The synaptic inputs rsuggest that gamma cells in rabbit retina would have light responses like their homologs in mouse retina, OFF responses to small stimuli in the receptive field center that are suppressed by a variety of larger stimuli. Thus, they would signal object motion selectively.

Predicting Age-related Changes with High Accuracy using a Pattern Recognition Derived Retinal Ganglion Cell Regression Model

This abstract was presented yesterday, May 7th at the 2017 Association for Research in Vision and Opthalmology (ARVO) meetings in Baltimore, Maryland by Nayuta Yoshioka, Barbara Zangerl, Lisa Nivison-Smith, Sieu Khuu, Bryan W. Jones, Rebecca Pfeiffer, Robert Marc, and Michael Kalloniatis.

Purpose: We recently used pattern recognition analysis to show macula areas can be classified into statistically distinct clusters in accordance to their age-related retinal ganglion cell layer (RGCL) thickness change in a normal population. The aim of this study was to perform a retrospective cross-sectional analysis utilizing a large cohort of patients to establish accuracy of this model and to develop a normative dataset using a 50-year-old equivalent cohort.

Methods: Data was collected from patients seen at the Centre for Eye Health for optic nerve assessment without posterior pole disease. The grid-wise RGCL thickness was obtained from a single eye of each patient via Spectralis OCT macular scan over an 8×8 measurement grid. Measurements for patients outside the 45-54 age range (training cohort) were converted to 50-year-old equivalent value utilizing pattern recognition derived regression model which, in brief, consists of 8×8 grid clustered into 8 distinct classes according to the pattern of RGCL thickness change with age. Accuracy of the predictions was assessed by comparing the training cohort’s measurements to the 45-54 year reference cohort using t-test and one-way ANOVA.

Results: Data were collected from a total 248 patients aged 20 to 78.1 years. 80 patients within this group were aged 45 – 54 and formed the reference cohort (average±SD 49.6±2.83) and the remaining 168 eyes formed the training cohort (average age±SD 50.7±17.34). Converted values for the training set matched those of the reference cohort (average disparity±SD 0.10±0.42µm, range -0.74-1.34µm) and were not significantly different (p > 0.9). Most variability was observed with patients above 70 years of age (average disparity±SD -0.09±1.73µm, range -3.67 to 6.16µm) and central grids corresponding to the fovea (average disparity±SD 0.47±0.72µm, range -0.22 to 1.34µm).

Conclusions: Our regression model for normal age-related RGCL change can accurately convert and/or predict RGCL thickness for individuals in comparison to 50-year-equivalent reference cohort and could allow for more accurate assessment of RGCL thickness and earlier detection of significant loss in the future. Caution may be needed when applying the model in the foveal area or for patients older than 70 years.

Metabolic Impacts of Cigarette Smoke On The Retina of Complement-Compromised Mice

This abstract was presented today, May 8th at the 2017 Association for Research in Vision and Opthalmology (ARVO) meetings in Baltimore, Maryland by Felix Vazquez-Chona, Alex Butler, Emile McKinnon, Baerbel Rohrer, and Bryan W. Jones. Full resolution version here.

Purpose: The interaction between metabolism and the immune system is hypothesized as playing a central role in the pathology of neural diseases including Age-Related Macular Degeneration (AMD). We investigated the effects of cigarette-smoke exposure (CSE) on metabolism of retinal cells in wild-type (wt) mice, and mice deficient for the alternative pathway (complement factor B, CfB) or common terminal pathway (complement component 3, C3).

Methods: Mice were exposed to CSE or room filtered-air (controls) for 6 h/d, 5 d/wk for 6 months. We visualized the metabolism of retinal cells using Computational Molecular Phenotyping (CMP). Retinal cell classification and metabolic adaptation were interrogated using arginine (R), aspartate (D), GABA (γ), glutamate (E), glycine (G), glutathione (J), glutamine (Q), taurine (τ), glutamine synthetase (GS), and cellular retinaldehyde binding protein (CRALBP). Electron microscope mosaics were instrumental in phenotyping metabolic profiles.

Results: CSE C3-/- animals show more severe degenerative indices than CSE WT: retinal pigment epithelium (RPE) exhibited a decreased basalateral infolding area and increased vacuolization; photoreceptors show increased mitochondrial swelling and pyknosis; Müller glia displayed hypertrophy; and the amacrine layer was affected by increased vacuolization. The CfB-/- retina was more resilient to the negative effects of CSE when compared to the WT retina. At the metabolic level, RPE and inner segments of CSE CfB-/- mice displayed modest changes. In contrast, changes in CSE C3-/- and WT retina were dramatic: RPE exhibited decreased CRALBP and elevated R-E-J-τ-γ levels; inner segments showed increased R-D-E-G-J-Q-τ-γ-CRALBP; and Müller glia were found to have decreased levels along the R-D-E-G-J-Q-τ-γ-GS-CRALBP axis.

Conclusions: Increased GABA levels in RPE and photoreceptors are consistent with Müller glia dysfunction. Our metabolic profiling suggests that RPE and Müller glia are vulnerable to CSE-induced oxidative stress. We also find that the potential complement activation status of the retina-RPE-choroid unit highly influences the metabolic response of retinal cells to CSE. As complete blockade of the complement system in the C3-/- model has a more dramatic impact on metabolism of RPE, Müller glia, and photoreceptors than observed in the CfB-/- model, it can be proposed that downstream signaling of the complement system is required for retina health.

Layman Abstract: Metabolism involves a complex circuitry of metabolic pathways, intermediates, and cell-cell interactions. Thus, mapping metabolism with cellular resolution and quantitative power is key to identifying robust biomarkers of disease progression.