Biology subjects

Anderson, M. G.

Publications and source records attributed to Anderson, M. G..

6 recordsLinked to original sources

bioRxiv2021

Tyr is Responsible for the Cctq1a QTL and Links Developmental Environment to Central Corneal Thickness Determination

Central corneal thickness is a quantitative trait with important associations to human health. In a phenotype-driven approach studying corneal thickness of congenic derivatives of C57BLKS/J and SJL/J mice, the critical region for a quantitative trait locus influencing corneal thickness, Cctq1a, was delimited to a 10-gene interval. Exome sequencing, RNAseq, and studying independent mutations eliminated multiple candidate genes and confirmed one. Though the causative gene, Tyr, has no obvious direct function in the transparent cornea, studies with multiple alleles on matched genetic backgrounds, both in isolation and genetic complementation crosses, confirmed allelism of Tyr-Cctq1a; albino mice lacking Tyr function had thin corneas. Albino mice also had increased axial length. Because albinism exposes eyes to increased light, the effect of dark-rearing was tested and found to rescue central corneal thickness. In sum, the results point to an epiphenomenon; developmental light exposure interacts with genotype as an important determinate of adult corneal thickness.

genetics↗

bioRxiv2021

AxonDeep: Automated Optic Nerve Axon Segmentation in Mice with Deep Learning.

PurposeOptic nerve damage is the principal feature of glaucoma and contributes to vision loss in many diseases. In animal models, nerve health has traditionally been assessed by human experts that grade damage qualitatively or manually quantify axons from sampling limited areas from histologic cross sections of nerve. Both approaches are prone to variability and are time consuming. First-generation automated approaches have begun to emerge, but all have significant shortcomings. Here, we seek improvements through use of deep-learning approaches for segmenting and quantifying axons from cross sections of mouse optic nerve. MethodsTwo deep-learning approaches were developed and evaluated: (1) a traditional supervised approach using a fully convolutional network trained with only labeled data and (2) a semi-supervised approach trained with both labeled and unlabeled data using a generative-adversarial-network framework. ResultsFrom comparisons with an independent test set of images with manually marked axon centers and boundaries, both deep-learning approaches outperformed an existing baseline automated approach and similarly to two independent experts. Performance of the semi-supervised approach was superior and implemented into AxonDeep. ConclusionAxonDeep performs automated quantification and segmentation of axons from healthy appearing nerves, and those with mild to moderate degrees of damage, similar to that of experts without the variability and constraints associated with manual performance. Translational RelevanceUse of deep learning for axon quantification provides rapid, objective, and higher throughput analysis of optic nerve that would otherwise not be possible.

bioengineering↗

bioRxiv2021

Quantification and image-derived phenotyping of retinal ganglion cell nuclei in the nee mouse model of congenital glaucoma

The nee mouse model exhibits characteristic features of congenital glaucoma, a common cause of childhood blindness. The current study of nee mice had two components. First, the time course of neurodegeneration in nee retinal flat-mounts was studied over time using a retinal ganglion cell (RGC)-marker, BRN3A; a pan-nuclear marker, TO-PRO-3; and H&E staining. Based on segmentation of nuclei using ImageJ and RetFM-J, this analysis identified a rapid loss of BRN3A+ nuclei from 4-15 weeks of age, with the first statistically significant difference in average density compared to age-matched controls detected in 8-week-old cohorts (49% reduction in nee). Consistent with a model of glaucoma, no reductions in BRN3A- nuclei were detected, but the combined analysis indicated that some RGCs lost BRN3A marker expression prior to actual cell loss. These results have a practical application in the design of experiments using nee mice to study mechanisms or potential therapies for congenital glaucoma. The second component of the study pertains to a discovery-based analysis of the large amount of image data with 748,782 segmented retinal nuclei. Using the automatedly collected region of interest feature data captured by ImageJ, we tested whether RGC density of glaucomatous mice was significantly correlated to average nuclear area, perimeter, Feret diameter, or MinFeret diameter. These results pointed to two events influencing nuclear size. For variations in RGC density above approximately 3,000 nuclei/mm2 apparent spreading was observed, in which BRN3A- nuclei--regardless of genotype--became slightly larger as RGC density decreased. This same spreading occurred in BRN3A+ nuclei of wild-type mice. For variation in RGC density below 3,000 nuclei/mm2, which only occurred in glaucomatous nee mutants, BRN3A+ nuclei became smaller as disease was progressively severe. These observations have relevance to defining RGCs of relatively higher sensitivity to glaucomatous cell death and the nuclear dynamics occurring during their demise.

molecular biology↗

bioRxiv2021

Recombinant adenovirus causes prolonged mobilization of macrophages in the anterior chamber of mice

PurposeOcular tissues of mice have been studied in many ways using replication deficient species C type 5 adenoviruses (Ad5) as tools for manipulating gene expression. While refinements to injection protocols and tropism have led to several advances in targeting cells of interest, there remains a relative lack of information concerning how Ad5 may influence other ocular cell types capable of confounding experimental interpretation. Here, a slit-lamp is used to thoroughly photodocument the sequelae of intraocular Ad5 injections over time in mice, with attention to potentially confounding indices of inflammation. MethodsA cohort of C57BL/6J mice was randomly split into 3 groups (Virus, receiving unilateral intracameral injection with 5x107 pfu of a cargo-less Ad5 construct; Saline, receiving unilateral balanced salt solution injection; and Naive, receiving no injections). From this initial experiment, a total of 52 eyes from 26 mice were photodocumented via slit-lamp at four time points (baseline, 1, 3, and 10 weeks following initiation of the experiment) by an observer masked to treatments and other parameters of the experimental design. Following the last in vivo exam, tissues were collected. Based on the slit-lamp data, tissues were studied via immunostaining with the macrophage marker F4/80. Subsequently, three iterations of the original experiment were performed with otherwise identical experimental parameters testing the effect of age, intravitreal injection, and A195 buffer, adding slit-lamp photodocumentation of an additional 32 eyes from 16 mice. ResultsThe masked investigator was able to use the sequential images from each mouse in the initial experiment to assign each mouse into its correct treatment group with near perfect fidelity. Virus injected eyes were characterized by corneal damage indicative of intraocular injection and a prolonged mobilization of clump cells on the surface of the iris. Saline injected eyes had only transient corneal opacities indicative of intraocular injections, and Naive eyes remained normal. Immunostaining with F4/80 was consistent with ascribing the clump cells visualized via slit-lamp imaging as a type of macrophage. Experimental iterations using Ad5 indicate that all virus injected eyes had the distinguishing feature of a prolonged presence of clump cells on the surface of the iris regardless of injection site. Mice receiving an intraocular injection of Ad5 at an advanced age displayed a protracted course of corneal cloudiness that prevented detailed visualization of the iris at the last time point. ConclusionsBecause the eye is often considered an "immune privileged site", we suspect that several studies have neglected to consider that the presence of Ad5 in the eye might evoke strong reactions from the innate immune system. Ad5 injection caused a sustained mobilization of clump cells, i.e. macrophages. This change is likely a consequence of either direct macrophage transduction or a secondary response to cytokines produced locally by other transduced cells. Regardless of how these cells were altered, the important implication is that the adenovirus led to long lasting changes in the environment of the anterior chamber. Thus, these findings describe a caveat of Ad5-mediated studies involving macrophage mobilization, which we encourage groups to use as a bioassay in their experiments and consider in interpretation of their ongoing experiments using adenoviruses.

physiology↗

bioRxiv2020

Biological Correlations and Confounding Variables for Quantification of Retinal Ganglion Cells Based on Optical Coherence Tomography Using Diversity Outbred Mice

PurposeDespite popularity of optical coherence tomography (OCT) in glaucoma studies, its unclear how well OCT-derived metrics compare to traditional measures of retinal ganglion cell (RGC) abundance. Here, Diversity Outbred (J:DO) mice are used to directly compare ganglion cell complex (GCC)-thickness measured by OCT to metrics of retinal anatomy measured ex vivo with retinal wholemounts or optic nerve cross sections. MethodsJ:DO mice (n = 48) underwent OCT and fundoscopic exams, with GCC-thickness measured using automated segmentation. Following euthanasia, RGC axons were quantified from para-phenylenediamine-stained optic nerve cross sections and RGC somas from BRN3A-immunolabeled retinal wholemounts with total cellularity assessed by TO-PRO or hematoxylin nuclear staining. ResultsJ:DO tissues lacked overt disease. GCC-thickness (62.4 {+/-} 3.7 {micro}m), RGC abundance (3,097 {+/-} 515 BRN3A+ nuclei/mm2; 45,533 {+/-} 9,077 axons), and total inner retinal cell abundance (6,952 {+/-} 810 nuclei/mm2) varied broadly. GCC-thickness correlated significantly to RGC somal density (r = 0.46) and axon number (r = 0.49), whereas total inner retinal cellularity did not. Retinal area (20.3 {+/-} 2.4 mm2) and optic nerve (0.09 {+/-} 0.02 mm2) cross-sectional area varied widely. Sex did not significantly influence any of these metrics. In bilateral comparisons, GCC-thickness (r = 0.89), inner retinal cellularity (r = 0.47), and RGC axon abundance (r = 0.72) all correlated significantly. ConclusionsAmongst outbred mice with widely variable phenotypes, OCT-derived measurements of GCC thickness correlate significantly to RGC abundance and axon number. The extensive phenotypic variability exhibited by J:DO mice make them a powerful resource for studies of retinal anatomy using quantitative genetics.

neuroscience↗

bioRxiv2019

Modulation of post-traumatic immune response using anti-IL-1 therapy for improved visual outcomes

The purpose of this study was to characterize acute changes in inflammatory pathways in the mouse eye following a blast-mediated TBI (bTBI) model, and to determine if modulation of these pathways could protect the structure and function of retinal ganglion cells (RGC). bTBI was induced in C57BL/6J male mice by exposure to three 20 PSI blast waves, with an interblast interval of one hour. Acute cytokine expression in retinal tissue was measured through real-time quantitative polymerase chain reaction (RT-qPCR) 4 hours post-blast. Increased retinal expression of lL-1{beta}, IL-1, IL-6, and TNF was observed in bTBI mice exposed to blast when compared to shams, which was associated with activation of microglia and macroglia reactivity, assessed via immunohistochemistry with IBA-1 and GFAP, respectively, 1 week post-blast. Inhibition of the IL-1 pathway was accomplished using anakina, an IL-1RI antagonist. Retinal function and structure were evaluated 4 weeks post-injury using pattern electroretinogram (PERG) and optical coherence tomography (OCT), respectively. After bTBI, anakinra treatment resulted in a preservation of RGC function and RGC structure when compared to saline treated bTBI mice. Optic nerve integrity analysis demonstrated a tred of decreased suggesting that IL-1 blockade also prevents axonal damage after blast. Blast exposure results in increased retinal inflammation including upregulation of pro-inflammatory cytokines and activation of resident microglia and macroglia. This may partially explain the RGC loss we observed in this model as blockade of the acute inflammatory response after injury with the IL-1R1 antagonist anakinra resulted in preservation of RGC function and structure.\n\nSignificance StatementBlast-mediated traumatic brain injury (bTBI) affects military members and civilians as a direct result of combat, workplace accidents, or intentional terrorist attacks. The retina is a central nervous system (CNS) tissue that is vulnerable to blast exposure. Individuals with bTBI often report visual dysfunction, but the mechanisms of ocular injury are poorly understood. This study demonstrates that damaging neuroinflammation contributes to retinal injury following blast-mediated TBI. We also identified anakinra, an anti-IL-1 therapy currently utilized for other diseases, as a potential pharmacologic agent that could prevent ocular damage after blast. These findings will aide in the development of novel treatments for vision preservation.

neuroscience↗