Visualizing the Human Body Using an Artistic Approach.

This chapter describes an innovative approach to the cross-disciplinary study of anatomy and art to facilitate visualization of the human body. We draw upon the literature, together with our own experience of designing, delivering and researching a cross-disciplinary art and anatomy course, to indicate the critical elements of the approach that foster students' visualization of the anatomy of the human body.Visual arts have been linked with anatomy for centuries, but typically biomedical science has existed in a utilitarian relationship with art only used as an aid. In this chapter, we discuss the rationale underpinning a cross-disciplinary anatomy and art course and describe our experience of devising activities and assessment that create a stimulating and mutually beneficial environment for visualizing the experience and physicality of the human body. We describe the structure of the course which integrates art and anatomy to train students in the language of anatomy and visual representation, by engaging them in a process of attempting their own visual communication. The cross-disciplinary nature of our approach creates a unique social environment that offers a supportive environment for exploration and experimentation without fear of failure. Students' personal growth in resilience, tolerance for uncertainty and creativity prepares them for the inclusion of these values in their career.

Comparison of 2 open-sourced 3-dimensional modeling techniques for orthopaedic application.

Objectives: Although 3-dimensional (3D) printing is becoming more widely adopted for clinical applications, it is yet to be accepted as part of standard practice. One of the key applications of this technology is orthopaedic surgical planning for urgent trauma cases. Anatomically accurate replicas of patients' fracture models can be produced to guide intervention. These high-quality models facilitate the design and printing of patient-specific implants and surgical devices. Therefore, a fast and accurate workflow will help orthopaedic surgeons to generate high-quality 3D printable models of complex fractures. Currently, there is a lack of access to an uncomplicated and inexpensive workflow. Methods: Using patient DICOM data sets (n = 13), we devised a novel, simple, open-source, and rapid modeling process using Drishti software and compared its efficacy and data storage with the 3D Slicer image computing platform. We imported the computed tomography image directory acquired from patients into the software to isolate the model of bone surface from surrounding soft tissue using the minimum functions. One pelvic fracture case was further integrated into the customized implant design practice to demonstrate the compatibility of the 3D models generated from Drishti. Results: The data sizes of the generated 3D models and the processing files that represent the original DICOM of Drishti are on average 27% and 12% smaller than that of 3D Slicer, respectively (both P < 0.05). The time frame needed to reach the stage of viewing the 3D bone model and the exporting of the data of Drishti is 39% and 38% faster than that of 3D Slicer, respectively (both P < 0.05). We also constructed a virtual model using third-party software to trial the implant design. Conclusions: Drishti is more suitable for urgent trauma cases that require fast and efficient 3D bone reconstruction with less hardware requirement. 3D Slicer performs better at quantitative preoperative planning and multilayer segmentation. Both software platforms are compatible with third-party programs used to produce customized implants that could be useful for surgical training. Level of Evidence: Level V.

Technology enhanced assessment: Ottawa consensus statement and recommendations.

INTRODUCTION: In 2011, a consensus report was produced on technology-enhanced assessment (TEA), its good practices, and future perspectives. Since then, technological advances have enabled innovative practices and tools that have revolutionised how learners are assessed. In this updated consensus, we bring together the potential of technology and the ultimate goals of assessment on learner attainment, faculty development, and improved healthcare practices. METHODS: As a material for the report, we used the scholarly publications on TEA in both HPE and general higher education, feedback from 2020 Ottawa Conference workshops, and scholarly publications on assessment technology practices during the Covid-19 pandemic. RESULTS AND CONCLUSION: The group identified areas of consensus that remained to be resolved and issues that arose in the evolution of TEA. We adopted a three-stage approach (readiness to adopt technology, application of assessment technology, and evaluation/dissemination). The application stage adopted an assessment 'lifecycle' approach and targeted five key foci: (1) Advancing authenticity of assessment, (2) Engaging learners with assessment, (3) Enhancing design and scheduling, (4) Optimising assessment delivery and recording learner achievement, and (5) Tracking learner progress and faculty activity and thereby supporting longitudinal learning and continuous assessment.

The question of dissection in medical training: Not just "if," but "when"? A student perspective.

While debate about the use of-and alternatives to-human cadaveric dissection in medical training is robust, little attention has been paid to questions about timing. This study explores the perspectives of medical students and recent graduates with regard to two key questions: when in the degree program do students prefer dissection opportunities and what are the students getting out of participating in dissection? Self-report survey data from students in preclinical years (n = 105), clinical years (n = 57), and graduates (n = 13) were analyzed. Most (89%) preferred dissection during the preclinical years, with no effect by training year (χ2  = 1.98, p = 0.16), previous anatomy (χ2  = 3.64, p = 0.31), or dissection (χ2  = 3.84, p = 0.26) experience. Three key findings emerged. First, the majority of students prefer to dissect in the preclinical years because they view dissection as important for developing foundation knowledge and delivering an opportunity for consolidation prior to transitioning to primarily clinical studies. In addition, students recognize that it is a time-consuming activity requiring specialized facilities. Second, three main understandings of the purpose of dissection were reported: depth of learning, learning experience, and real-world equivalence. Third, these student perspectives of the purpose of dissection are associated with timing preferences for dissection opportunities. The results identify the preclinical phase as the optimal time to strategically integrate dissection into medical training in order to maximize the benefits of this unique learning opportunity for students and minimize its impact upon curricular time.

Examining the Short-, Medium-, and Long-Term Success of an Embodied Learning Activity in the Study of Hand Anatomy for Clinical Application.

A student's own body provides an often disregarded site of knowledge production and corporeal wisdom. Learning via cognitive processes anchored in physical movement and body awareness, known as embodied learning, may aid students to visualize structures and understand their functions and clinical relevance. Working from an embodied learning perspective, the current article evaluates the use of an offline physical learning tool (Anatomical Glove Learning System; AGLS) for teaching hand anatomy for clinical application in medical students. Two student samples (N1  = 105; N2  = 94) used the AGLS in two different ways. In the first sample, the AGLS was compared to a traditional approach using hand bones, models and prosected specimens. Secondly, the AGLS and traditional approach were combined. The evaluation consisted of three outcomes: short-term learning (post-test), medium-term applications (mock-objective structured clinical examination, MOSCE), and longer-term assessment (objective structured clinical examination, OSCE). Findings from the first sample indicated no significant differences between the AGLS and traditional laboratory groups on short- (F(1,78) = 0.036, P = 0.849), medium- (F(1,50) = 0.743, P = 0.393), or longer-term (F(1,82) = 0.997, P = 0.321) outcomes. In the second sample using the AGLS in combination with a traditional approach was associated with significantly better short-term post-test scores (F(2,174) = 5.98, P = 0.003) than using the AGLS alone, but demonstrated no effect for long-term OSCE scores. These results suggest an embodied learning experience alone does not appear to be advantageous to student learning, but when combined with other methods for studying anatomy there are learning gains.

Forced Disruption of Anatomy Education in Australia and New Zealand: An Acute Response to the Covid-19 Pandemic.

Australian and New Zealand universities commenced a new academic year in February/March 2020 largely with "business as usual." The subsequent Covid-19 pandemic imposed unexpected disruptions to anatomical educational practice. Rapid change occurred due to government-imposed physical distancing regulations from March 2020 that increasingly restricted anatomy laboratory teaching practices. Anatomy educators in both these countries were mobilized to adjust their teaching approaches. This study on anatomy education disruption at pandemic onset within Australia and New Zealand adopts a social constructivist lens. The research question was "What are the perceived disruptions and changes made to anatomy education in Australia and New Zealand during the initial period of the Covid-19 pandemic, as reflected on by anatomy educators?." Thematic analysis to elucidate "the what and why" of anatomy education was applied to these reflections. About 18 anatomy academics from ten institutions participated in this exercise. The analysis revealed loss of integrated "hands-on" experiences, and impacts on workload, traditional roles, students, pedagogy, and anatomists' personal educational philosophies. The key opportunities recognized for anatomy education included: enabling synchronous teaching across remote sites, expanding offerings into the remote learning space, and embracing new pedagogies. In managing anatomy education's transition in response to the pandemic, six critical elements were identified: community care, clear communications, clarified expectations, constructive alignment, community of practice, ability to compromise, and adapt and continuity planning. There is no doubt that anatomy education has stepped into a yet unknown future in the island countries of Australia and New Zealand.

A method for gene knockdown in the retina using a lipid-based carrier.

Purpose: The use of small non-coding nucleic acids, such as siRNA and miRNA, has allowed for a deeper understanding of gene functions, as well as for development of gene therapies for complex neurodegenerative diseases, including retinal degeneration. For effective delivery into the eye and transfection of the retina, suitable transfection methods are required. We investigated the use of a lipid-based transfection agent, Invivofectamine® 3.0 (Thermo Fisher Scientific), as a potential method for delivery of nucleic acids to the retina. Methods: Rodents were injected intravitreally with formulations of Invivofectamine 3.0 containing scrambled, Gapdh, Il-1ß, and C3 siRNAs, or sterile PBS (control) using a modified protocol for encapsulation of nucleic acids. TdT-mediated dUTP nick-end labeling (TUNEL) and IBA1 immunohistochemistry was used to determine histological cell death and inflammation. qPCR were used to determine the stress and inflammatory profile of the retina. Electroretinography (ERG) and optical coherence tomography (OCT) were employed as clinical indicators of retinal health. Results: We showed that macrophage recruitment, retinal stress, and photoreceptor cell death in animals receiving Invivofectamine 3.0 were comparable to those in negative controls. Following delivery of Invivofectamine 3.0 alone, no statistically significant changes in expression were found in a suite of inflammatory and stress genes, and ERG and OCT analyses revealed no changes in retinal function or morphology. Injections with siRNAs for proinflammatory genes (C3 and Il-1ß) and Gapdh, in combination with Invivofectamine 3.0, resulted in statistically significant targeted gene knockdown in the retina for up to 4 days following injection. Using a fluorescent Block-It siRNA, transfection was visualized throughout the neural retina with evidence of transfection observed in cells of the ganglion cell layer, inner nuclear layer, and outer nuclear layer. Conclusions: This work supports the use of Invivofectamine 3.0 as a transfection agent for effective delivery of nucleic acids to the retina for gene function studies and as potential therapeutics.

How Can We Show You, If You Can't See It? Trialing the Use of an Interactive Three-Dimensional Micro-CT Model in Medical Education.

Teaching internal structures obscured from direct view is a major challenge of anatomy education. High-fidelity interactive three-dimensional (3D) micro-computed tomography (CT) models with virtual dissection present a possible solution. However, their utility for teaching complex internal structures of the human body is unclear. The purpose of this study was to investigate the use of a realistic 3D micro-CT interactive visualization computer model to teach paranasal sinus anatomy in a laboratory setting during pre-clinical medical training. Year 1 (n = 79) and Year 2 (n = 59) medical students undertook self-directed activities focused on paranasal sinus anatomy in one of two laboratories (traditional laboratory and 3D model). All participants completed pre and posttests before and after the laboratory session. Results of regression analyses predicting post-laboratory knowledge indicate that, when students were inexperienced with the 3D computer technology, use of the model was detrimental to learning for students with greater prior knowledge of the relevant anatomy (P < 0.05). For participants experienced with the 3D computer technology, however, the use of the model was detrimental for students with less prior knowledge of the relevant anatomy (P < 0.001). These results emphasize that several factors need to be considered in the design and effective implementation of such models in the classroom. Under the right conditions, the 3D model is equal to traditional laboratory resources when used as a learning tool. This paper discusses the importance of preparatory training for students and the technical consideration necessary to successfully integrate such models into medical anatomical curricula.

Sex, but not skin tone affects penetration of red-light (660 nm) through sites susceptible to sports injury in lean live and cadaveric tissues.

Red-light treatment is emerging as a novel therapy for promoting tissue recovery but data on red-light penetration through human tissues are lacking. We aimed to: (1) determine the effect of light irradiance, tissue thickness, skin tone, sex and bone/muscle content on 660 nm light penetration through common sites of sports injuries, and (2) establish if cadaver tissues serve as a useful model for predicting red-light penetration in live tissues. Live and cadaver human tissues were exposed to 660 nm light at locations across the skull, spinal cord and upper and lower limbs. Red-light was produced by a light emitting diode array of various irradiances (15-500 mW/cm2 ) and measured by a light-probe positioned on the tissue surface opposite to the light emitting diodes. 100 mW/cm2 successfully penetrated tissue <50 mm thick; a disproportionate irradiance increase was required to achieve deeper penetration. Penetration was unaffected by skin tone, increased with irradiance and relative bone/muscle composition, and decreased with greater tissue thickness and in males. Live and cadaveric tissue penetration did not differ statistically for tissues <50 mm but cadavers required more red-light to penetrate >50 mm. These results assist clinicians and researchers in determining red-light treatment intensities for penetrating human tissues.

Photoreceptor Survival Is Regulated by GSTO1-1 in the Degenerating Retina.

Purpose: Glutathione-S-transferase omega 1-1 (GSTO1-1) is a cytosolic glutathione transferase enzyme, involved in glutathionylation, toll-like receptor signaling, and calcium channel regulation. GSTO1-1 dysregulation has been implicated in oxidative stress and inflammation, and contributes to the pathogenesis of several diseases and neurological disorders; however, its role in retinal degenerations is unknown. The aim of this study was to investigate the role of GSTO1-1 in modulating oxidative stress and consequent inflammation in the normal and degenerating retina. Methods: The role of GSTO1-1 in retinal degenerations was explored by using Gsto1-/- mice in a model of retinal degeneration. The expression and localization of GSTO1-1 were investigated with immunohistochemistry and Western blot. Changes in the expression of inflammatory (Ccl2, Il-1ß, and C3) and oxidative stress (Nox1, Sod2, Gpx3, Hmox1, Nrf2, and Nqo1) genes were investigated via quantitative real-time polymerase chain reaction. Retinal function in Gsto1-/- mice was investigated by using electroretinography. Results: GSTO1-1 was localized to the inner segment of cone photoreceptors in the retina. Gsto1-/- photo-oxidative damage (PD) mice had decreased photoreceptor cell death as well as decreased expression of inflammatory (Ccl2, Il-1ß, and C3) markers and oxidative stress marker Nqo1. Further, retinal function in the Gsto1-/- PD mice was increased as compared to wild-type PD mice. Conclusions: These results indicate that GSTO1-1 is required for inflammatory-mediated photoreceptor death in retinal degenerations. Targeting GSTO1-1 may be a useful strategy to reduce oxidative stress and inflammation and ameliorate photoreceptor loss, slowing the progression of retinal degenerations.

MicroRNA-124 Dysregulation is Associated With Retinal Inflammation and Photoreceptor Death in the Degenerating Retina.

Purpose: We sought to determine the role and retinal cellular location of microRNA-124 (miR-124) in a neuroinflammatory model of retinal degeneration. Further, we explored the anti-inflammatory relationship of miR-124 with a predicted messenger RNA (mRNA) binding partner, chemokine (C-C motif) ligand 2 (Ccl2), which is crucially involved in inflammatory cell recruitment in the damaged retina. Methods: Human AMD donor eyes and photo-oxidative damaged (PD) mice were labeled for miR-124 expression using in situ hybridization. PDGFRa-cre RFP mice were used for Müller cell isolation from whole retinas. MIO-M1 immortalized cells and rat primary Müller cells were used for in vitro analysis of miR-124 expression and its relationship with Ccl2. Therapeutic efficacy was tested with intravitreal administration of miR-124 mimic in mice, with electroretinography used to determine retinal function. IBA1 immunohistochemistry and photoreceptor row counts were used for assessment of inflammation and cell death. Results: MiR-124 expression was correlated with progressive retinal damage, inflammation, and cell death in human AMD and PD mice. In addition, miR-124 expression was inversely correlated to Ccl2 expression in mice following PD. MiR-124 was localized to both neuronal-like photoreceptors and glial (Müller) cells in the retina, with a redistribution from neurons to glia occurring as a consequence of PD. Finally, intravitreal administration of miR-124 mimics decreased retinal inflammation and photoreceptor cell death, and improved retinal function. Conclusions: This study has provided an understanding of the mechanism behind miR-124 in the degenerating retina and demonstrates the usefulness of miR-124 mimics for the modulation of retinal degenerations.

Dynamic Interplay of Innate and Adaptive Immunity During Sterile Retinal Inflammation: Insights From the Transcriptome.

The pathogenesis of many retinal degenerations, such as age-related macular degeneration (AMD), is punctuated by an ill-defined network of sterile inflammatory responses. The delineation of innate and adaptive immune milieu among the broad leukocyte infiltrate, and the gene networks, which construct these responses, are poorly described in the eye. Using photo-oxidative damage in a rodent model of subretinal inflammation, we employed a novel RNA-sequencing framework to map the global gene network signature of retinal leukocytes. This revealed a previously uncharted interplay of adaptive immunity during subretinal inflammation, including prolonged enrichment of myeloid and lymphocyte migration, antigen presentation, and the alternative arm of the complement cascade involving Factor B. We demonstrate Factor B-deficient mice are protected against macrophage infiltration and subretinal inflammation. Suppressing the drivers of retinal leukocyte proliferation, or their capacity to elicit complement responses, may help preserve retinal structure and function during sterile inflammation in diseases such as AMD.

Obesity-induced metabolic disturbance drives oxidative stress and complement activation in the retinal environment.

Purpose: Systemic increases in reactive oxygen species, and their association with inflammation, have been proposed as an underlying mechanism linking obesity and age-related macular degeneration (AMD). Studies have found increased levels of oxidative stress biomarkers and inflammatory cytokines in obese individuals; however, the correlation between obesity and retinal inflammation has yet to be assessed. We used the leptin-deficient (ob/ob) mouse to further our understanding of the contribution of obesity to retinal oxidative stress and inflammation. Methods: Retinas from ob/ob mice were compared to age-matched wild-type controls for retinal function (electroretinography) and gene expression analysis of retinal stress (Gfap), oxidative stress (Gpx3 and Hmox1), and complement activation (C3, C2, Cfb, and Cfh). Oxidative stress was further quantified using a reactive oxygen species and reactive nitrogen species (ROS and RNS) assay. Retinal microglia and macrophage migration to the outer retina and complement activation were determined using immunohistochemistry for IBA1 and C3, respectively. Retinas and sera were used for metabolomic analysis using QTRAP mass spectrometry. Results: Retinal function was reduced in ob/ob mice, which correlated to changes in markers of retinal stress, oxidative stress, and inflammation. An increase in C3-expressing microglia and macrophages was detected in the outer retinas of the ob/ob mice, while gene expression studies showed increases in the complement activators (C2 and Cfb) and a decrease in a complement regulator (Cfh). The expression of several metabolites were altered in the ob/ob mice compared to the controls, with changes in polyunsaturated fatty acids (PUFAs) and branched-chain amino acids (BCAAs) detected. Conclusions: The results of this study indicate that oxidative stress, inflammation, complement activation, and lipid metabolites in the retinal environment are linked with obesity in ob/ob animals. Understanding the interplay between these components in the retina in obesity will help inform risk factor analysis for acquired retinal degenerations, including AMD.

The use of the vaccinia virus complement control protein (VCP) in the rat retina.

The complement system is highly implicated in both the prevalence and progression of Age-Related Macular Degeneration (AMD). Complement system inhibitors therefore have potential therapeutic value in managing excessive activation of the complement pathways in retinal degenerations. The vaccinia virus complement control protein (VCP) has been shown to be effective as a complement inhibitor in neuroinflammatory models including traumatic brain injury and spinal cord injury. We aimed to investigate the potential of VCP as a therapeutic molecule for retinal degenerations. In this study, we investigated the effect, localisation and delivery of VCP to the rodent retina. Complement inhibition activity of VCP was tested using a hemolytic assay. Photoreceptor cell death, inflammation and retinal stress were assayed to determine if any retinal toxicity was induced by an intravitreal injection of VCP. The effect of VCP was investigated in a model of photo-oxidative retinal degeneration. Localisation of VCP after injection was determined using a fluorescein-tagged form of VCP, as well as immunohistochemistry. Finally, a copolymer resin (Elvax) was trialled for the slow-release delivery of VCP to the retina. We found that a dose equivalent to 20µg VCP when intravitreally injected into the rat eye did not cause any photoreceptor cell death or immune cell recruitment, but led to an increase in GFAP. In photo-oxidative damaged retinas, there were no differences in photoreceptor loss, retinal stress (Gfap) and inflammation (Ccl2 and C3) between VCP and saline-injected groups; however, Jun expression was reduced in VCP-treated retinas. After VCP was injected into the eye, it was taken up in all layers of the retina but was cleared within 1-3 hours of delivery. This study indicates that a method to sustain the delivery of VCP to the retina is necessary to further investigate the effect of VCP as a complement inhibitor for retinal degenerations.

670nm light treatment following retinal injury modulates Müller cell gliosis: Evidence from in vivo and in vitro stress models.

Photobiomodulation (PBM) with 670 nm light has been shown to accelerate wound healing in soft tissue injuries, and also to protect neuronal tissues. However, little data exist on its effects on the non-neuronal components of the retina, such as Müller cells (MCs), which are the principal macroglia of the retina that play a role in maintaining retinal homeostasis. The aim of this study was to explore the effects of 670 nm light on activated MCs using in vivo and in vitro stress models. Adult Sprague-Dawley rats were exposed to photo-oxidative damage (PD) for 24 h and treated with 670 nm light at 0, 3 and 14 days after PD. Tissue was collected at 30 days post-PD for analysis. Using the in vitro scratch model with a human MC line (MIO-M1), area coverage and cellular stress were analysed following treatment with 670 nm light. We showed that early treatment with 670 nm light after PD reduced MC activation, lowering the retinal expression of GFAP and FGF-2. 670 nm light treatment mitigated the production of MC-related pro-inflammatory cytokines (including IL-1ß), and reduced microglia/macrophage (MG/MΦ) recruitment into the outer retina following PD. This subsequently decreased photoreceptor loss, slowing the progression of retinal degeneration. In vitro, we showed that 670 nm light directly modulated MC activation, reducing rates of area coverage by suppressing cellular proliferation and spreading. This study indicates that 670 nm light treatment post-injury may have therapeutic benefit when administered shortly after retinal damage, and could be useful for retinal degenerations where MC gliosis is a feature of disease progression.

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain.

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

Photobiomodulation with 670 nm light ameliorates Müller cell-mediated activation of microglia and macrophages in retinal degeneration.

Müller cells, the supporting cells of the retina, play a key role in responding to retinal stress by releasing chemokines, including CCL2, to recruit microglia and macrophages (MG/MΦ) into the damaged retina. Photobiomodulation (PBM) with 670 nm light has been shown to reduce inflammation in models of retinal degeneration. In this study, we aimed to investigate whether 670 nm light had an effect on Müller cell-initiated inflammation under retinal photo-oxidative damage (PD) in vivo and in vitro. Sprague-Dawley rats were pre-treated with 670 nm light (9J/cm2) once daily over 5 days prior to PD. The expression of inflammatory genes including CCL2 and IL-1ß was analysed in retinas. In vitro, primary Müller cells dissociated from neonatal rat retinas were co-cultured with 661W photoreceptor cells. Co-cultures were exposed to PD, followed by 670 nm light treatment to the Müller cells only, and Müller cell stress and inflammation were assessed. Primary MG/MΦ were incubated with supernatant from the co-cultures, and collected for analysis of inflammatory activation. To further understand the mechanism of 670 nm light, the expression of COX5a and mitochondrial membrane potential (ΔΨm) were measured in Müller cells. Following PD, 670 nm light-treated Müller cells had a reduced inflammatory activation, with lower levels of CCL2, IL-1ß and IL-6. Supernatant from 670 nm light-treated co-cultures reduced activation of primary MG/MΦ, and lowered the expression of pro-inflammatory cytokines, compared to untreated PD controls. Additionally, 670 nm light-treated Müller cells had an increased expression of COX5a and an elevated ΔΨm following PD, suggesting that retrograde signaling plays a role in the effects of 670 nm light on Müller cell gene expression. Our data indicates that 670 nm light reduces Müller cell-mediated retinal inflammation, and offers a potential cellular mechanism for 670 nm light therapy in regulating inflammation associated with retinal degenerations.

Retinal metabolic events in preconditioning light stress as revealed by wide-spectrum targeted metabolomics.

INTRODUCTION: Light is the primary stimulus for vision, but may also cause damage to the retina. Pre-exposing the retina to sub-lethal amount of light (or preconditioning) improves chances for retinal cells to survive acute damaging light stress. OBJECTIVES: This study aims at exploring the changes in retinal metabolome after mild light stress and identifying mechanisms that may be involved in preconditioning. METHODS: Retinas from 12 rats exposed to mild light stress (1000 lux × for 12 h) and 12 controls were collected one and seven days after light stress (LS). One retina was used for targeted metabolomics analysis using the Biocrates p180 kit while the fellow retina was used for histological and immunohistochemistry analysis. RESULTS: Immunohistochemistry confirmed that in this experiment, a mild LS with retinal immune response and minimal photoreceptor loss occurred. Compared to controls, LS induced an increased concentration in phosphatidylcholines. The concentration in some amino acids and biogenic amines, particularly those related to the nitric oxide pathway (like asymmetric dimethylarginine (ADMA), arginine and citrulline) also increased 1 day after LS. 7 days after LS, the concentration in two sphingomyelins and phenylethylamine was found to be higher. We further found that in controls, retina metabolome was different between males and females: male retinas had an increased concentration in tyrosine, acetyl-ornithine, phosphatidylcholines and (acyl)-carnitines. CONCLUSIONS: Besides retinal sexual metabolic dimorphism, this study shows that preconditioning is mostly associated with re-organisation of lipid metabolism and changes in amino acid composition, likely reflecting the involvement of arginine-dependent NO signalling.

Retinal Macrophages Synthesize C3 and Activate Complement in AMD and in Models of Focal Retinal Degeneration.

Purpose: Complement system dysregulation is strongly linked to the progression of age-related macular degeneration (AMD). Deposition of complement including C3 within the lesions in atrophic AMD is thought to contribute to lesion growth, although the contribution of local cellular sources remains unclear. We investigated the role of retinal microglia and macrophages in complement activation within atrophic lesions, in AMD and in models of focal retinal degeneration. Methods: Human AMD donor retinas were labeled for C3 expression via in situ hybridization. Rats were subject to photo-oxidative damage, and lesion expansion was tracked over a 2-month period using optical coherence tomography (OCT). Three strategies were used to determine the contribution of local and systemic C3 in mice: total C3 genetic ablation, local C3 inhibition using intravitreally injected small interfering RNA (siRNA), and depletion of serum C3 using cobra venom factor. Results: Retinal C3 was expressed by microglia/macrophages located in the outer retina in AMD eyes. In rodent photo-oxidative damage, C3-expressing microglia/macrophages and complement activation were located in regions of lesion expansion in the outer retina over 2 months. Total genetic ablation of C3 ameliorated degeneration and complement activation in retinas following damage, although systemic depletion of serum complement had no effect. In contrast, local suppression of C3 expression using siRNA inhibited complement activation and deposition, and reduced cell death. Conclusions: These findings implicate C3, produced locally by retinal microglia/macrophages, as contributing causally to retinal degeneration. Consequently, this suggests that C3-targeted gene therapy may prove valuable in slowing the progression of AMD.

Microglia-derived IL-1ß promotes chemokine expression by Müller cells and RPE in focal retinal degeneration.

BACKGROUND: Chemokine signalling is required for the homing of leukocytes during retinal inflammation, and is associated with pathogenesis of diseases such as age-related macular degeneration (AMD). Here, we explore the role of interleukin-1ß (IL-1ß) in modulating AMD-associated chemokines Ccl2, Cxcl1, and Cxcl10 during photo-oxidative retinal damage, and the effect on both the accumulation of outer-retinal macrophages, and death of photoreceptors. METHODS: Inhibition of retinal IL-1ß expression was performed using either siRNA or antibody neutralisation, which was intravitreally injected in SD rats prior to photo-oxidative damage. Changes in the expression and localisation of Il-1ß, Ccl2, Cxcl1 and Cxcl10 genes were assessed using qPCR and in situ hybridisation, while the recruitment of retinal macrophages was detected using immunohistochemistry for IBA1. Levels of photoreceptor cell death were determined using TUNEL. RESULTS: Photo-oxidative damage elevated the expression of Il-1ß and inflammasome-related genes, and IL-1ß protein was detected in microglia infiltrating the outer retina. This was associated with increased expression of Ccl2, Cxcl1, and Cxcl10. Intravitreal IL-1ß inhibitors suppressed chemokine expression following damage and reduced macrophage accumulation and photoreceptor death. Moreover, in Müller and RPE cell cultures, and in vivo, Ccl2, Cxcl1 and Cxcl10 were variously upregulated when stimulated with IL-1ß, with increased macrophage accumulation detected in vivo. CONCLUSIONS: IL-1ß is produced by retinal microglia and macrophages and promotes chemokine expression by Müller cells and RPE in retinal degeneration. Targeting IL-1ß may prove efficacious in broadly suppressing chemokine-mediated inflammation in retinal dystrophies such as AMD.