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The composition and location of professional antigen presenting cells (APC) varies in different mucosal surfaces. The cornea, long considered an immune-privileged tissue devoid of APCs, is now known to host a heterogeneous network of bone marrow-derived cells. Here, we utilized transgenic mice that express enhanced green fluorescent protein (EGFP) from the CD11c promoter (pCD11c) in conjunction with immunohistochemical staining to demonstrate an interesting stratification of APCs within non-inflamed murine corneas. pCD11c+ dendritic cells (DCs) reside in the basal epithelium, seemingly embedded in the basement membrane. Most DCs express MHC class II on at least some dendrites, which extend up to 50 μm in length and traverse up 20 μm tangentially towards the apical surface of the epithelium. The DC density diminishes from peripheral to central cornea. Beneath the DCs and adjacent to the stromal side of the basement membrane reside pCD11c-CD11b+ putative macrophages that express low levels of MHC class II. Finally, MHC class II-pCD11c-CD11b+ cells form a network throughout the remainder of the stroma. This highly reproducible stratification of bone marrow-derived cells is suggestive of a progression from an APC function at the exposed corneal surface to an innate immune barrier function deeper in the stroma. [ABSTRACT FROM AUTHOR]

Aim: To describe advances in 3D data capture and printing that allow photorealistic replicas of human anatomical specimens for education and research, and discuss advantages of current generation printing for replica design and manufacture. Materials & methods: We combine surface scanning and computerized tomography datasets that maximize precise color and geometric capture with ultra violet (UV) curable resin printing to replicate human anatomical specimens. Results: We describe the process for color control, print design and translation of photorealistic 3D meshes into 3D prints in durable resins. Conclusion: Current technologies allow previously unachievable ability to capture and reproduce anatomical specimens, and provide a platform for a new generation of 3D printed teaching materials to be designed and used in anatomy education environments.

Capturing the 'third dimension' of complex human form or anatomy has been an objective of artists and anatomists from the renaissance in the fifteenth and sixteenth centuries onwards. Many of these drawings, paintings, and sculptures have had a profound influence on medical teaching and the learning resources we took for granted until around 40 years ago. Since then, the teaching of human anatomy has undergone significant change, especially in respect of the technologies available to augment or replace traditional cadaver-based dissection instruction. Whilst resources such as atlases, wall charts, plastic models, and images from the Internet have been around for many decades, institutions looking to reduce the reliance on dissection-based teaching in medical or health professional training programmes have in more recent times increasingly had access to a range of other options for classroom-based instruction. These include digital resources and software programmes and plastinated specimens, although the latter come with a range of ethical and cost considerations. However, the urge to recapitulate the 'third dimension' of anatomy has seen the recent advent of novel resources in the form of 3D printed replicas. These 3D printed replicas of normal human anatomy dissections are based on a combination of radiographic imaging and surface scanning that captures critical 3D anatomical information. The final 3D files can either be augmented with false colour or made to closely resemble traditional prosections prior to printing. This chapter details the journey we and others have taken in the search for the 'third dimension'. The future of a haptically identical, anatomically accurate replica of human cadaver specimens for surgical and medical training is nearly upon us. Indeed, the need for hard copy replicas may eventually be superseded by the opportunities afforded by virtual reality (VR) and augmented reality (AR)., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Background: Pediatric airway models currently available for use in education or simulation do not replicate anatomy or tissue responses to procedures. Emphasis on mass production with sturdy but homogeneous materials and low-fidelity casting techniques diminishes these models' abilities to realistically represent the unique characteristics of the pediatric airway, particularly in the infant and younger age ranges. Newer fabrication technologies, including 3-dimensional (3D) printing and castable tissue-like silicones, open new approaches to the simulation of pediatric airways with greater anatomical fidelity and utility for procedure training., Methods: After ethics approval, available/archived computerized tomography data sets of patients under the age of 2 years were reviewed to identify those suitable for designing new models. A single 21-month-old subject was selected for 3D reconstruction. Manual thresholding was then performed to produce 3D models of selected regions and tissue types within the dataset, which were either directly 3D-printed or later cast in 3D-printed molds with a variety of tissue-like silicones. A series of testing mannequins derived using this multimodal approach were then further refined following direct clinician feedback to develop a series of pediatric airway model prototypes., Results: The initial prototype consisted of separate skeletal (skull, mandible, vertebrae) and soft-tissue (nasal mucosa, pharynx, larynx, gingivae, tongue, functional temporomandibular joint [TMJ] "sleeve," skin) modules. The first iterations of these modules were generated using both single-material and multimaterial 3D printing techniques to achieve the haptic properties of real human tissues. After direct clinical feedback, subsequent prototypes relied on a combination of 3D printing for osseous elements and casting of soft-tissue components from 3D-printed molds, which refined the haptic properties of the nasal, oropharyngeal, laryngeal, and airway tissues, and improved the range of movement required for airway management procedures. This approach of modification based on clinical feedback resulted in superior functional performance., Conclusions: Our hybrid manufacturing approach, merging 3D-printed components and 3D-printed molds for silicone casting, allows a more accurate representation of both the anatomy and functional characteristics of the pediatric airway for model production. Further, it allows for the direct translation of anatomy derived from real patient medical imaging into a functional airway management simulator, and our modular design allows for modification of individual elements to easily vary anatomical configurations, haptic qualities of components or exchange components to replicate pathology., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Anesthesia Research Society.)

Purpose: In spite of clear differences in tissue function and significance to ocular disease, little is known about how immune responses differ between the retina and uveal tract. To this end we compared the effects of acute systemic inflammation on myeloid cells within the mouse retina, iris-ciliary body, and choroid., Methods: Systemic inflammation was induced in Cx3cr1gfp/gfp and CD11c-eYFP Crb1wt/wtmice by intraperitoneal lipopolysaccharide (LPS). In vivo fundus imaging was performed at two, 24, and 48 hours after LPS, and ocular tissue wholemounts were immunostained and studied by confocal microscopy. Flow cytometry was used to investigate the expression of activation markers (MHC class II, CD80, CD86) on myeloid cell populations at 24 hours. For functional studies, retinal microglia were isolated from LPS-exposed mice and cocultured with naïve OT-II CD4+ T-cells and ovalbumin peptide. T-cell proliferation was measured by flow cytometry and cytokine assays., Results: Systemic LPS altered the density and morphology of retinal microglia; however, retinal microglia did not upregulate antigen presentation markers and failed to stimulate naïve CD4+ T-cell proliferation in vitro. In contrast, uveal tract myeloid cells displayed a phenotype consistent with late-activated antigen-presenting cells at 24 hours. Systemic LPS induced remodeling of myeloid populations within the uveal tract, particularly in the choroid, where dendritic cells were partially displaced by macrophages at 24 hours., Conclusions: The disparate myeloid cell responses in the retina and uveal tract after systemic LPS highlight differential regulation of innate immunity within these tissue environments, observations that underpin and advance our understanding of ocular immune privilege.

Purpose: Given the role of corneal sensory nerves during epithelial wound repair, we sought to examine the relationship between immune cells and polymodal nociceptors following corneal injury., Methods: Young C57BL/6J mice received a 2 mm corneal epithelial injury. One week later, corneal wholemounts were immunostained using β-tubulin-488, TRPV1 (transient receptor potential ion channel subfamily V member-1, a nonselective cation channel) and immune cell (MHC-II, CD45 and CD68) antibodies. The sum length of TRPV1+ and TRPV1- nerve fibers, and their spatial association with immune cells, was quantified in intact and injured corneas., Results: TRPV1+ nerves account for ∼40% of the nerve fiber length in the intact corneal epithelium and ∼80% in the stroma. In the superficial epithelial layers, TRPV1+ nerve terminal length was similar in injured and intact corneas. In intact corneas, the density (sum length) of basal epithelial TRPV1+ and TRPV1- nerve fibers was similar, however, in injured corneas, TRPV1+ nerve density was higher compared to TRPV1- nerves. The degree of physical association between TRPV1+ nerves and intraepithelial CD45+ MHC-II+ CD11c+ cells was similar in intact and injured corneas. Stromal leukocytes co-expressed TRPV1, which was partially localized to CD68+ lysosomes, and this expression pattern was lower in injured corneas., Conclusions: TRPV1+ nerves accounted for a higher proportion of corneal nerves after injury, which may provide insights into the pathophysiology of neuropathic pain following corneal trauma. The close interactions of TRPV1+ nerves with intraepithelial immune cells and expression of TRPV1 by stromal macrophages provide evidence of neuroimmune interactions in the cornea.

The teaching of medical pathology has undergone significant change in the last 30-40 years, especially in the context of employing bottled specimens or 'pots' in classroom settings. The reduction in post-mortem based teaching in medical training programs has resulted in less focus being placed on the ability of students to describe the gross anatomical pathology of specimens. Financial considerations involved in employing staff to maintain bottled specimens, space constraints and concerns with health and safety of staff and student laboratories have meant that many institutions have decommissioned their pathology collections. This report details how full-colour surface scanning coupled with CT scanning and 3 D printing allows the digital archiving of gross pathological specimens and the production of reproductions or replicas of preserved human anatomical pathology specimens that obviates many of the above issues. With modern UV curable resin printing technology, it is possible to achieve photographic quality accurate replicas comparable to the original specimens in many aspects except haptic quality. Accurate 3 D reproductions of human pathology specimens offer many advantages over traditional bottled specimens including the capacity to generate multiple copies and their use in any educational setting giving access to a broader range of potential learners and users.

Purpose: We report novel differences in mouse corneal DC morphology and density during local and systemic inflammation., Methods: Local inflammation was induced by topical application of saline or TLR9 agonist CpG-ODN on abraded C57BL6J mouse corneas. Systemic inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS) in CD11c-YFP mice. Corneal epithelial DCs from uninjured, injured and contralateral eyes were analysed by confocal microscopy., Results: Following local CpG delivery on the injured cornea, the DC density and size increased in both central and peripheral regions. Contralateral uninjured eyes displayed enlarged DC morphology in the central cornea compared to naïve cohorts. After systemic LPS, the size of DCs in the central cornea was lower at 2 hours, returning to baseline after 24 hours., Conclusions: Corneal DCs respond differently in terms of shape and distribution during local and systemic inflammation. These features can serve as in vivo indicators in ocular and systemic diseases.

Purpose: Human choroidal melanocytes become evident in the last trimester of development, but very little is known about them. To better understand normal and diseased choroidal melanocyte biology we examined their precursors, melanoblasts (MB), in mouse eyes during development, particularly their relation to the developing vasculature and immune cells., Methods: Naïve B6(Cg)-Tyrc-2J/J albino mice were used between embryonic (E) day 15.5 and postnatal (P) day 8, with adult controls. Whole eyes, posterior segments, or dissected choroidal wholemounts were stained with antibodies against tyrosinase-related protein 2, ionized calcium binding adaptor molecule-1 or isolectin B4, and examined by confocal microscopy. Immunoreactive cell numbers in the choroid were quantified with Imaris. One-way ANOVA with Tukey's post hoc test assessed statistical significance., Results: Small numbers of MB were present in the presumptive choroid at E15.5 and E18.5. The density significantly increased between E18.5 (381.4 ± 45.8 cells/mm2) and P0 (695.2 ± 87.1 cells/mm2; P = 0.032). In postnatal eyes MB increased in density and formed multiple layers beneath the choriocapillaris. MB in the periocular mesenchyme preceded the appearance of vascular structures at E15.5. Myeloid cells (Ionized calcium binding adaptor molecule-1-positive) were also present at high densities from this time, and attained adult-equivalent densities by P8 (556.4 ± 73.6 cells/mm2)., Conclusions: We demonstrate that choroidal MB and myeloid cells are both present at very early stages of mouse eye development (E15.5). Although MB and vascularization seemed to be unlinked early in choroidal development, they were closely associated at later stages. MB did not migrate into the choroid in waves, nor did they have a consistent relationship with nerves.

There is accumulating evidence that aging shifts the central nervous system milieu towards a proinflammatory state, with increased reactivity of microglia in the aging eye and brain having been implicated in the development of age-related neurodegenerative conditions. Indeed, alterations to microglial morphology and function have been recognized as a part of normal aging. Here, we sought to assess the effects of age on the retinal microglial and macrophage response to acute intraocular pressure (IOP) elevation. Further, we performed experiments whereby bone marrow from young or middle-aged mice was used to reconstitute the bone marrow of whole-body irradiated 12 month old mice. Bone marrow chimeric mice then underwent cannulation and IOP elevation 8 weeks after whole-body irradiation and bone marrow transplantation in order to determine whether the age of bone marrow alters the macrophage response to retinal injury. Our data show retinal macrophage reactivity and microglial morphological changes were enhanced in older mice when compared to younger mice in response to injury. When IOP elevation was performed after whole-body irradiation and bone marrow rescue, we noted subretinal macrophage accumulation and glial reactivity was reduced compared to non-irradiated mice that had also undergone IOP elevation. This effect was evident in both groups of chimeric mice that had received either young or middle-aged bone marrow, suggesting irradiation itself may alter the macrophage and glial response to injury rather than the age of bone marrow., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Previous studies have reported that topical exposure to the toll-like receptor (TLR) 9 ligand CpG-ODN causes widespread ocular inflammation, including retinal microglial activation and posterior segment inflammation. Here we sought to determine the effects of systemic exposure to CpG-ODN in the retina and whether this inflammatory response was altered with Cx 3 cr1 deficiency or hyperglycemia. Male non-diabetic Cx 3 cr1 +/gfp and Cx 3 cr1 gfp/gfp littermates (normoglycemic controls) and Cx 3 cr1 +/gfp Ins2 Akita and Cx 3 cr1 gfp/gfp Ins2 Akita diabetic mice were injected intraperitoneally with 40 μg CpG-ODN. Immunofluorescence staining was performed 1 week later to assess the expression of MHC Class II and glial fibrillary acidic protein (GFAP), as well as to identify morphological changes to microglia and changes in retinal macrophage cell density. Systemic exposure to CpG-ODN induced the upregulated expression of both GFAP on retinal Müller cells and MHC Class II on the retinal vasculature. Additionally, there was an increased accumulation of macrophages in the subretinal space 1 week after exposure to systemic CpG-ODN as well as characteristic morphological changes to microglia indicative of an activated phenotype. These preliminary studies demonstrate that low-grade inflammatory changes were not enhanced in Cx 3 cr1-deficient or diabetic mice, indicating that the inflammatory response to systemic CpG-ODN in the retina is unaltered in the context of Cx 3 cr1 deficiency or prolonged hyperglycemia., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Neurosurgical residents are finding it more difficult to obtain experience as the primary operator in aneurysm surgery. The present study aimed to replicate patient-derived cranial anatomy, pathology and human tissue properties relevant to cerebral aneurysm intervention through 3D printing and 3D print-driven casting techniques. The final simulator was designed to provide accurate simulation of a human head with a middle cerebral artery (MCA) aneurysm., Methods: This study utilized living human and cadaver-derived medical imaging data including CT angiography and MRI scans. Computer-aided design (CAD) models and pre-existing computational 3D models were also incorporated in the development of the simulator. The design was based on including anatomical components vital to the surgery of MCA aneurysms while focusing on reproducibility, adaptability and functionality of the simulator. Various methods of 3D printing were utilized for the direct development of anatomical replicas and moulds for casting components that optimized the bio-mimicry and mechanical properties of human tissues. Synthetic materials including various types of silicone and ballistics gelatin were cast in these moulds. A novel technique utilizing water-soluble wax and silicone was used to establish hollow patient-derived cerebrovascular models., Results: A patient-derived 3D aneurysm model was constructed for a MCA aneurysm. Multiple cerebral aneurysm models, patient-derived and CAD, were replicated as hollow high-fidelity models. The final assembled simulator integrated six anatomical components relevant to the treatment of cerebral aneurysms of the Circle of Willis in the left cerebral hemisphere. These included models of the cerebral vasculature, cranial nerves, brain, meninges, skull and skin. The cerebral circulation was modeled through the patient-derived vasculature within the brain model. Linear and volumetric measurements of specific physical modular components were repeated, averaged and compared to the original 3D meshes generated from the medical imaging data. Calculation of the concordance correlation coefficient (ρ c : 90.2%-99.0%) and percentage difference (≤0.4%) confirmed the accuracy of the models., Conclusions: A multi-disciplinary approach involving 3D printing and casting techniques was used to successfully construct a multi-component cerebral aneurysm surgery simulator. Further study is planned to demonstrate the educational value of the proposed simulator for neurosurgery residents.

In the eye immune defenses must take place in a plethora of differing microenvironments ranging from the corneal and conjunctival epithelia facing the external environment to the pigmented connective tissue of the uveal tract containing smooth muscle, blood vessels and peripheral nerves to the innermost and highly protected neural retina. The extravascular environment of the neural retina, like the brain parenchyma, is stringently controlled to maintain conditions required for neural transmission. The unique physiological nature of the neural retina can be attributed to the blood retinal barriers (BRB) of the retinal vasculature and the retinal pigment epithelium, which both tightly regulate the transport of small molecules and restrict passage of cells and macromolecules from the circulation into the retina in a similar fashion to the blood brain barrier (BBB). The extracellular environment of the neural retina differs markedly from that of the highly vascular, loose connective tissue of the choroid, which lies outside the BRB. The choroid hosts a variety of immune cell types, including macrophages, dendritic cells (DCs) and mast cells. This is in marked contrast to the neural parenchyma of the retina, which is populated almost solely by microglia. This review will describe the current understanding of the distribution, phenotype and physiological role of ocular immune cells behind or inside the blood-retinal barriers and those in closely juxtaposed tissues outside the barrier. The nature and function of these immune cells can profoundly influence retinal homeostasis and lead to disordered immune function that can lead to vision loss., (Copyright © 2018. Published by Elsevier Ltd.)

The central nervous system (CNS) is considered to be immune privileged, owing in part to the absence of major histocompatibility (MHC) class II + cells in the healthy brain parenchyma. However, systemic inflammation can activate microglia to express MHC class II, suggesting that systemic inflammation may be sufficient to mature microglia into functional antigen presenting cells (APCs). We examined the effects of systemic lipopolysaccharide (LPS)-induced inflammation on the phenotype and function of putative APCs within the mouse brain parenchyma, as well as its supporting tissues-the choroid plexus and meninges. Microglia isolated from different regions of the brain demonstrated significant heterogeneity in their ability to present antigen to naïve OT-II CD4 + T cells following exposure to systemic LPS. Olfactory bulb microglia (but not cortical microglia) intimately interacted with T cells in vivo and stimulated T cell proliferation in vitro, albeit in the absence of co-stimulation. In contrast, myeloid cells within the choroid plexus and meninges were immunogenic and upregulated the co-stimulatory molecule CD80 following systemic inflammation. Dural APCs, which clustered around LYVE-1 + lymphatics, were more efficient at stimulating naïve T cell proliferation than choroid plexus APCs, suggesting that the dura may be an under-appreciated site for immune interactions. This study has highlighted the functional diversity of myeloid cells within the sub-compartments of the CNS and its supporting tissues. Furthermore, these findings demonstrate that systemic inflammation can mature selected microglia populations and choroid plexus/meningeal myeloid cells into functional APCs, which may contribute to the pathogenesis of neuroinflammation and neurodegenerative diseases., (© 2018 Wiley Periodicals, Inc.)

The practical aspect of human developmental biology education is often limited to the observation and use of animal models to illustrate developmental anatomy. This is due in part to the difficulty of accessing human embryonic and fetal specimens, and the sensitivity inherent to presenting these specimens as teaching materials. This report presents a new approach using three-dimensional (3D) printed replicas of actual human materials in practical classes, thus allowing for the inclusion of accurate examples of human developmental anatomy in the educational context. A series of 3D prints have been produced from digital data collected by computed tomography (CT) imaging of an archived series of preserved human embryonic and fetal specimens. The final versions of 3D prints have been generated in a range of single or multiple materials to illustrate the progression of human development, including the development of internal anatomy. Furthermore, multiple copies of each replica have been printed for large group teaching. In addition to the educational benefit of examining accurate 3D replicas, this approach lessens the potential for adverse student reaction (due to cultural background or personal experience) to observing actual human embryonic/fetal anatomical specimens, and reduces the potential of damage or loss of original specimens. This approach, in combination with ongoing improvements in the management and analysis of digital data and advances in scanning technology, has enormous potential to allow embryology students access to both local and international collections of human gestational material. Anat Sci Educ 00: 000-000. © 2018 American Association of Anatomists., (© 2018 American Association of Anatomists.)

Classically, the CNS is described as displaying immune privilege, as it shows attenuated responses to challenge by alloantigen. However, the CNS does show local inflammation in response to infection. Although pathogen access to the brain parenchyma and retina is generally restricted by physiological and immunological barriers, certain pathogens may breach these barriers. In the CNS, such pathogens may either cause devastating inflammation or benefit from immune privilege in the CNS, where they are largely protected from the peripheral immune system. Thus, some pathogens can persist as latent infections and later be reactivated. We review the consequences of immune privilege in the context of CNS infections and ask whether immune privilege may provide protection for certain pathogens and promote their latency.

With the availability of numerous adjuncts or alternatives to learning anatomy other than cadavers (medical imaging, models, body painting, interactive media, virtual reality) and the costs of maintaining cadaver laboratories, it was considered timely to have a mature debate about the need for cadavers in the teaching of undergraduate medicine. This may be particularly pertinent given the exponential growth in medical knowledge in other disciplines, which gives them valid justification for time in already busy medical curricula. In this symposium, the pros and cons of cadaver use in modern medical curricula were debated and audience participation encouraged.

Objectives: Rapid prototyping (RP) technology is becoming more affordable, faster, and is now capable of building models with a high resolution and accuracy. Due to technological limitations, 3D printing in biological anthropology has been mostly limited to museum displays and forensic reconstructions. In this study, we compared the accuracy of different 3D printers to establish whether RP can be used effectively to reproduce anthropological dental collections, potentially replacing access to oftentimes fragile and irreplaceable original material., Methods: We digitized specimens from the Yuendumu collection of Australian Aboriginal dental casts using a high-resolution white-light scanning system and reproduced them using four different 3D printing technologies: stereolithography (SLA); fused deposition modeling (FDM); binder-jetting; and material-jetting. We compared the deviations between the original 3D surface models with 3D print scans using color maps generated from a 3D metric deviation analysis., Results: The 3D printed models reproduced both the detail and discrete morphology of the scanned dental casts. The results of the metric deviation analysis demonstrate that all 3D print models were accurate, with only a few small areas of high deviations. The material-jetting and SLA printers were found to perform better than the other two printing machines., Conclusions: The quality of current commercial 3D printers has reached a good level of accuracy and detail reproduction. However, the costs and printing times limit its application to produce large sample numbers for use in most anthropological studies. Nonetheless, RP offers a viable option to preserve numerically constraint fragile skeletal and dental material in paleoanthropological collections., (© 2018 Wiley Periodicals, Inc.)

Serine/threonine kinase 35 (STK35) is a recently identified human kinase with an autophosphorylation function, linked functionally to actin stress fibers, cell cycle progression and survival. STK35 has previously been shown to be highly expressed in human testis, and we demonstrated its regulation by nuclear-localized importin α2 in HeLa cells. The present study identifies progressive expression from the STK35 locus of two coding mRNA isoforms and one long non-coding RNA (lncRNA) in mouse testis during spermatogenesis, indicating their tightly controlled synthesis. Additionally, lncRNA transcripts are increased by exposure to oxidative stress in mouse GC-1 germ cell line. STK35 knockout (KO) mice lacking all three RNAs are born at sub-Mendelian frequency, and adults manifest both male and female germline deficiency. KO males exhibit no or partial spermatogenesis in most testis tubule cross-sections; KO ovaries are smaller and contain fewer follicles. Eyes of KO mice display phenotypes ranging from gross deformity to mild goniodysgenesis or iridocorneal angle malformation, to overtly normal. These findings demonstrate the tight regulation of transcription from the STK35 locus and its central importance to fertility, eye development and cell responses to oxidative stress., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Rapid progress is being made in understanding the roles of the cerebral meninges in the maintenance of normal brain function, in immune surveillance, and as a site of disease. Most basic research on the meninges and the neural brain is now done on mice, major attractions being the availability of reporter mice with fluorescent cells, and of a huge range of antibodies useful for immunocytochemistry and the characterization of isolated cells. In addition, two-photon microscopy through the unperforated calvaria allows intravital imaging of the undisturbed meninges with sub-micron resolution. The anatomy of the dorsal meninges of the mouse (and, indeed, of all mammals) differs considerably from that shown in many published diagrams: over cortical convexities, the outer layer, the dura, is usually thicker than the inner layer, the leptomeninx, and both layers are richly vascularized and innervated, and communicate with the lymphatic system. A membrane barrier separates them and, in disease, inflammation can be localized to one layer or the other, so experimentalists must be able to identify the compartment they are studying. Here, we present current knowledge of the functional anatomy of the meninges, particularly as it appears in intravital imaging, and review their role as a gateway between the brain, blood, and lymphatics, drawing on information that is scattered among works on different pathologies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In marsupials that possess a retinal vasculature, the arterial and venous segments, down to the smallest calibre capillaries, have been shown to occur in pairs. This pattern is seen in the marsupial central nervous system (CNS) but not in other tissues in this group or in any tissues in eutherian mammals. The present study aimed to determine if the gray short-tailed opossum (Monodelphis domestica), a south American marsupial, possesses double retinal vessels. Secondly, we investigated the relationship between vessels and astrocytes and microglia, which are known to play pivotal roles in the blood retinal barrier and immune surveillance respectively. Eyes from M. domestica between 2 months and 33 months of age were examined by bright field and fluorescein angiography, resin histology, and wholemount immunostaining. Retinal vessels in this marsupial always occur in closely related pairs with the arteriolar limb usually on the vitread aspect. Branches penetrate the retina to form layers of paired capillaries as far as the outer nuclear layer. Dense networks of GFAP + astrocytes enveloped the vitread aspect of vessels. No particularly strong association with blood vessels and ramified Iba1 + and Ib4 + microglia was noted. M. domestica possessed the unusual paired vasculature and capillary loops arrangement previously described in the marsupial CNS. These observations in a small laboratory-friendly marsupial open up new frontiers to investigate the factors that regulate paired blood vessel development and the functional significance of this arrangement when compared to the anastomotic pattern observed in the retina of eutherian mammals. Anat Rec, 300:1391-1400, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The eye is a complex sensory organ composed of a range of tissue types including epithelia, connective tissue, smooth muscle, vascular and neural tissue. While some components of the eye require a high level of transparency to allow light to pass through unobstructed, other tissues are characterized by their dense pigmentation, which functions to absorb light and thus control its passage through the ocular structures. Macrophages are present in all ocular tissues, from the cornea at the anterior surface through to the choroid/sclera at the posterior pole. This review will describe the current understanding of the distribution, phenotype, and physiological role of ocular macrophages, and provide a summary of evidence pertaining to their proposed role during pathological conditions.

Purpose: To describe a mouse model of hyperoxia-induced vitreoretinopathy that replicated some of the clinical and pathologic features encountered in infants with severe retinopathy of prematurity and congenital ocular conditions such as persistent hyperplastic primary vitreous., Methods: Experimental mice (C57BL/6J) were exposed to 65% oxygen between postnatal days (P)0 to P7 and studied at P10, P14, and 3, 5, 8, 20, and 40 weeks. Controls were exposed to normoxic conditions. Fundus imaging and fluorescein angiography were performed at all time points, and spectral-domain optical coherence tomography (SD-OCT) and electroretinography were performed at 8- and 20-week time points. Eyes were processed for resin histology, frozen sections, and retinal whole mounts. Immunostaining was performed to visualize vasculature isolectin B4 (Ib4), collagen type IV, glial fibrillary acidic protein, and α-smooth muscle actin., Results: Early exposure to hyperoxia resulted in bilateral vitreous hemorrhages at 3 weeks. From 5 weeks onward there were extensive zones of retinal degeneration, scarring or gliosis, retinal folding, and detachments caused by traction of α-smooth muscle actin-positive vitreous membranes. Tortuous retinal vessels, together with hyperplastic and persistence of hyaloid vessels are evident into adulthood. In the early stages (P10-3 weeks), branches from the tunica vasculosa lentis (TVL) supplied the marginal retina until retinal vessels were established. The peripheral retina remained poorly vascularized into adulthood. Electroretinography revealed 50% to 60% diminution in retinal function in adult mice that strongly correlated with vitreal changes identified using SD-OCT., Conclusions: This animal model displays a mixture of vitreoretinal pathologic changes that persist into adulthood. The model may prove valuable in experimental investigations of therapeutic approaches to blinding conditions caused by vitreous and retinal abnormalities.

To explore the potential viability and limitations of 3D printed models of children with cleft palate deformity., Background: The advantages of 3D printed replicas of normal anatomical specimens have previously been described. The creation of 3D prints displaying patient-specific anatomical pathology for surgical planning and interventions is an emerging field. Here we explored the possibility of taking rare pediatric radiographic data sets to create 3D prints for surgical education., Methods: Magnetic resonance imaging data of 2 children (8 and 14 months) were segmented, colored, and anonymized, and stereolothographic files were prepared for 3D printing on either multicolor plastic or powder 3D printers and multimaterial 3D printers., Results: Two models were deemed of sufficient quality and anatomical accuracy to print unamended. One data set was further manipulated digitally to artificially extend the length of the cleft. Thus, 3 models were printed: 1 incomplete soft-palate deformity, 1 incomplete anterior palate deformity, and 1 complete cleft palate. All had cleft lip deformity. The single-material 3D prints are of sufficient quality to accurately identify the nature and extent of the deformities. Multimaterial prints were subsequently created, which could be valuable in surgical training., Conclusion: Improvements in the quality and resolution of radiographic imaging combined with the advent of multicolor multiproperty printer technology will make it feasible in the near future to print 3D replicas in materials that mimic the mechanical properties and color of live human tissue making them potentially suitable for surgical training., Competing Interests: The authors have no financial interest to declare in relation to the content of this article. This study was supported by Monash University Centre for Human Anatomy Education—3D printing equipment and software. The Article Processing Charge was paid for by the authors.

Under steady-state conditions the central nervous system (CNS) is traditionally thought to be devoid of antigen presenting cells; however, putative dendritic cells (DCs) expressing enhanced yellow fluorescent protein (eYFP) are present in the retina and brain parenchyma of CD11c-eYFP mice. We previously showed that these mice carry the Crb1(rd8) mutation, which causes retinal dystrophic lesions; therefore we hypothesized that the presence of CD11c-eYFP(+) cells within the CNS may be due to pathology associated with the Crb1(rd8) mutation. We generated CD11c-eYFP Crb1(wt/wt) mice and compared the distribution and immunophenotype of CD11c-eYFP(+) cells in CD11c-eYFP mice with and without the Crb1(rd8) mutation. The number and distribution of CD11c-eYFP(+) cells in the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice. CD11c-eYFP(+) cells were distributed throughout the inner retina, and clustered in brain regions that receive input from the external environment or lack a blood-brain barrier. CD11c-eYFP(+) cells within the retina and cerebral cortex of CD11c-eYFP Crb1(wt/wt) mice expressed CD11b, F4/80, CD115 and Iba-1, but not DC or antigen presentation markers, whereas CD11c-eYFP(+) cells within the choroid plexus and pia mater expressed CD11c, I-A/I-E, CD80, CD86, CD103, DEC205, CD8α and CD135. The immunophenotype of CD11c-eYFP(+) cells and microglia within the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice; however, CD11c and I-A/I-E expression was significantly increased in CD11c-eYFP Crb1(rd8/rd8) mice. This study demonstrates that the overwhelming majority of CNS CD11c-eYFP(+) cells do not display the phenotype of DCs or their precursors and are most likely a subpopulation of microglia. GLIA 2016. GLIA 2016;64:1331-1349., (© 2016 Wiley Periodicals, Inc.)

Three-dimensional (3D) printing is an emerging technology capable of readily producing accurate anatomical models, however, evidence for the use of 3D prints in medical education remains limited. A study was performed to assess their effectiveness against cadaveric materials for learning external cardiac anatomy. A double blind randomized controlled trial was undertaken on undergraduate medical students without prior formal cardiac anatomy teaching. Following a pre-test examining baseline external cardiac anatomy knowledge, participants were randomly assigned to three groups who underwent self-directed learning sessions using either cadaveric materials, 3D prints, or a combination of cadaveric materials/3D prints (combined materials). Participants were then subjected to a post-test written by a third party. Fifty-two participants completed the trial; 18 using cadaveric materials, 16 using 3D models, and 18 using combined materials. Age and time since completion of high school were equally distributed between groups. Pre-test scores were not significantly different (P = 0.231), however, post-test scores were significantly higher for 3D prints group compared to the cadaveric materials or combined materials groups (mean of 60.83% vs. 44.81% and 44.62%, P = 0.010, adjusted P = 0.012). A significant improvement in test scores was detected for the 3D prints group (P = 0.003) but not for the other two groups. The finding of this pilot study suggests that use of 3D prints do not disadvantage students relative to cadaveric materials; maximally, results suggest that 3D may confer certain benefits to anatomy learning and supports their use and ongoing evaluation as supplements to cadaver-based curriculums. Anat Sci Educ 9: 213-221. © 2015 American Association of Anatomists., (© 2015 American Association of Anatomists.)

Granulocyte colony-stimulating factor (G-CSF) is a regulator of neutrophil production, function, and survival. Herein, we investigated the role of G-CSF in a murine model of human uveitis-experimental autoimmune uveoretinitis. Experimental autoimmune uveoretinitis was dramatically reduced in G-CSF-deficient mice and in anti-G-CSF monoclonal antibody-treated, wild-type (WT) mice. Flow cytometric analysis of the ocular infiltrate in WT mice with experimental autoimmune uveoretinitis showed a mixed population, comprising neutrophils, macrophages, and T cells. The eyes of G-CSF-deficient and anti-G-CSF monoclonal antibody-treated WT mice had minimal neutrophil infiltrate, but no change in other myeloid-derived inflammatory cells. Antigen-specific T-cell responses were maintained, but the differentiation of pathogenic type 17 helper T cells in experimental autoimmune uveoretinitis was reduced with G-CSF deficiency. We show that G-CSF controls the ocular neutrophil infiltrate by modulating the expression of C-X-C chemokine receptors 2 and 4 on peripheral blood neutrophils, as well as actin polymerization and migration. These data reveal an integral role for G-CSF-driven neutrophil responses in ocular autoimmunity, operating within and outside of the bone marrow, and also identify G-CSF as a potential therapeutic target in the treatment of human uveoretinitis., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: We previously have reported that application of the intracellular toll-like receptor (TLR)-9 ligand CpG-ODN onto the injured corneal surface induces widespread inflammation within the eye, including the retina. We tested the hypothesis that topical application of two other intracellular TLR agonists, Poly I:C and R848, would cause retinal microglial activation and migration into the subretinal space., Methods: C57BL/6J wild-type and Cx3cr1gfp/+ mice were anesthetized and received central corneal abrasions followed by topical application of Poly I:C (TLR3 agonist), R848 (TLR7/8 agonist), or CpG-ODN (TLR9 agonist). Eyes were imaged in vivo by using spectral-domain optical coherence tomography to assess and quantify vitreous cells and retinal edema. Tissues were processed for whole-mount immunofluorescence staining or gene expression studies. Microglial activation was determined by morphologic changes, major histocompatibility complex (MHC) class II reactivity, and migration to the subretinal space. Expression of proinflammatory cytokine gene IL-6, IL-1β, IFN-γ, and MCP-1 in retinal tissues were analyzed., Results: At 24 hours, topical treatment with CpG-ODN and R848, but not Poly I:C, led to altered microglial morphology. One week after CpG-ODN and R848-treatment, eyes exhibited vitritis and mild inner retinal edema, increased number of subretinal Iba-1+ cells, and an increase in MHC II+ cells in the neural retina. Proinflammatory cytokine genes were upregulated after R848 treatment, whereas in the CpG-ODN group, only IL-1β and MCP-1 were significantly upregulated. Retinal microglial activation was not observed in the Poly I:C-treated group., Conclusions: Topical application of CpG-ODN and R848, but not Poly I:C, to the damaged corneal surface can cause activation and migration of retinal microglia.

Background: The teaching of human head, neck and orbital anatomy forms a critical part of undergraduate and postgraduate medical and allied health professional training, including optometry. While still largely grounded in cadaveric dissection, this method of instruction is constrained in some countries and regional areas by access to real human cadavers, costs of cadaver bequest programmes, health and safety of students and staff and the shortage of adequate time in modern curricula. Many candidates choosing a postgraduate pathway in ophthalmological training, such as those accepted into the Royal Colleges of Ophthalmology in the UK, Australia and New Zealand programmes and the American Academy of Ophthalmologists in the USA, are compelled as adult learners to revise or revisit human orbital anatomy, ocular anatomy and select areas of head and neck anatomy. These candidates are often then faced with the issue of accessing facilities with dissected human cadaveric material., Methods: In light of these difficulties, we developed a novel means of creating high-resolution reproductions of prosected human cadaver orbits suitable for education and training., Results: 3D printed copies of cadaveric orbital dissections (superior, lateral and medial views) showing a range of anatomical features were created., Discussion: These 3D prints offer many advantages over plastinated specimens as they are suitable for rapid reproduction and as they are not human tissue they avoid cultural and ethical issues associated with viewing cadaver specimens. In addition, they are suitable for use in the office, home, laboratory or clinical setting in any part of the world for patient and doctor education., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

The mouse retina is a commonly used animal model for the study of pathogenesis and treatment of blinding retinal vascular diseases such as diabetic retinopathy. In this study, we aimed to characterize normal and pathological variations in vascular anatomy in the mouse retina using fluorescein angiography visualized with scanning laser ophthalmoscopy and optical coherence tomography (SLO-OCT). We examined eyes from C57BL/6J wild type mice as well as the Ins2(Akita) and Akimba mouse models of diabetic retinopathy using the Heidelberg Retinal Angiography (HRA) and OCT system. Angiography was performed on three focal planes to examine distinct vascular layers. For comparison with angiographic data, ex vivo analyses, including Indian ink angiography, histology and 3D confocal scanning laser microscopy were performed in parallel. All layers of the mouse retinal vasculature could be readily visualized during fluorescein angiography by SLO-OCT. Blood vessel density was increased in the deep vascular plexus (DVP) compared with the superficial vascular plexus (SVP). Arteriolar and venular typologies were established and structural differences were observed between venular types. Unexpectedly, the hyaloid artery was found to persist in 15% of C57BL/6 mice, forming anastomoses with peripheral retinal capillaries. Fluorescein leakage was easily detected in Akimba retinae by angiography, but was not observed in Ins2(Akita) mice. Blood vessel density was increased in the DVP of 6 month old Ins2(Akita) mice, while the SVP displayed reduced branching in precapillary arterioles. In summary, we present the first comprehensive characterization of the mouse retinal vasculature by SLO-OCT fluorescein angiography. Using this clinical imaging technique, we report previously unrecognized variations in C57BL/6J vascular anatomy and novel features of vascular retinopathy in the Ins2(Akita) mouse model of diabetes., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Modern imaging techniques are an essential component of preoperative planning in plastic and reconstructive surgery. However, conventional modalities, including three-dimensional (3D) reconstructions, are limited by their representation on 2D workstations. 3D printing, also known as rapid prototyping or additive manufacturing, was once the province of industry to fabricate models from a computer-aided design (CAD) in a layer-by-layer manner. The early adopters in clinical practice have embraced the medical imaging-guided 3D-printed biomodels for their ability to provide tactile feedback and a superior appreciation of visuospatial relationship between anatomical structures. With increasing accessibility, investigators are able to convert standard imaging data into a CAD file using various 3D reconstruction softwares and ultimately fabricate 3D models using 3D printing techniques, such as stereolithography, multijet modeling, selective laser sintering, binder jet technique, and fused deposition modeling. However, many clinicians have questioned whether the cost-to-benefit ratio justifies its ongoing use. The cost and size of 3D printers have rapidly decreased over the past decade in parallel with the expiration of key 3D printing patents. Significant improvements in clinical imaging and user-friendly 3D software have permitted computer-aided 3D modeling of anatomical structures and implants without outsourcing in many cases. These developments offer immense potential for the application of 3D printing at the bedside for a variety of clinical applications. In this review, existing uses of 3D printing in plastic surgery practice spanning the spectrum from templates for facial transplantation surgery through to the formation of bespoke craniofacial implants to optimize post-operative esthetics are described. Furthermore, we discuss the potential of 3D printing to become an essential office-based tool in plastic surgery to assist in preoperative planning, developing intraoperative guidance tools, teaching patients and surgical trainees, and producing patient-specific prosthetics in everyday surgical practice.

Background: Experimental autoimmune uveoretinitis (EAU) is a widely used experimental animal model of human endogenous posterior uveoretinitis. In the present study, we performed in vivo imaging of the retina in transgenic reporter mice to investigate dynamic changes in exogenous inflammatory cells and endogenous immune cells during the disease process., Methods: Transgenic mice (C57Bl/6 J Cx 3 cr1 (GFP/+) , C57Bl/6 N CD11c-eYFP, and C57Bl/6 J LysM-eGFP) were used to visualize the dynamic changes of myeloid-derived cells, putative dendritic cells and neutrophils during EAU. Transgenic mice were monitored with multi-modal fundus imaging camera over five time points following disease induction with the retinal auto-antigen, interphotoreceptor retinoid binding protein (IRBP1-20). Disease severity was quantified with both clinical and histopathological grading., Results: In the normal C57Bl/6 J Cx 3 cr1 (GFP/+) mouse Cx3cr1-expressing microglia were evenly distributed in the retina. In C57Bl/6 N CD11c-eYFP mice clusters of CD11c-expressing cells were noted in the retina and in C57Bl/6 J LysM-eGFP mice very low numbers of LysM-expressing neutrophils were observed in the fundus. Following immunization with IRBP1-20, fundus examination revealed accumulations of Cx3cr1-GFP(+) myeloid cells, CD11c-eYFP(+) cells and LysM-eGFP(+) myelomonocytic cells around the optic nerve head and along retinal vessels as early as day 14 post-immunization. CD11c-eYFP(+) cells appear to resolve marginally earlier (day 21 post-immunization) than Cx3cr1-GFP(+) and LysM-eGFP(+) cells. The clinical grading of EAU in transgenic mice correlated closely with histopathological grading., Conclusions: These results illustrate that in vivo fundus imaging of transgenic reporter mice allows direct visualization of various exogenously and endogenously derived leukocyte types during EAU progression. This approach acts as a valuable adjunct to other methods of studying the clinical course of EAU.

Macrophage adherence to the inner corneal surface and formation of MGCs in the stroma are common signs of chronic inflammation following corneal infection. To determine whether macrophage adherence (known clinically as KPs) and giant cell formation were specific to innate immune activation via particular TLR ligands, macrophage activation was examined in a murine model of TLR-mediated corneal inflammation. The corneal epithelium was debrided and highly purified TLR ligands were topically applied once to the cornea of TLR7(-/-), TLR9(-/-), Cx3cr1(gfp/+), CD11c(eYFP), and IL-4(-/-) mice. At 1 week post-treatment macrophage activation and phenotype was evaluated in the cornea. Treatment with TLR2, TLR3, TLR4, and TLR5 ligands caused an increase in the number of activated stromal macrophages in the central cornea at 1 week post-treatment. However, treatment with TLR9 ligand CpG-ODN and the TLR7/8 ligand R848/Resiquimod led to an accumulation of macrophages on the corneal endothelium and formation of multinucleated giant macrophages in the corneal stroma. We suggest that giant cell formation, which is a characteristic feature of granuloma formation in many tissues, may be a unique feature of TLR9- and TLR7/8-mediated macrophage activation., (© Society for Leukocyte Biology.)

The teaching of anatomy has consistently been the subject of societal controversy, especially in the context of employing cadaveric materials in professional medical and allied health professional training. The reduction in dissection-based teaching in medical and allied health professional training programs has been in part due to the financial considerations involved in maintaining bequest programs, accessing human cadavers and concerns with health and safety considerations for students and staff exposed to formalin-containing embalming fluids. This report details how additive manufacturing or three-dimensional (3D) printing allows the creation of reproductions of prosected human cadaver and other anatomical specimens that obviates many of the above issues. These 3D prints are high resolution, accurate color reproductions of prosections based on data acquired by surface scanning or CT imaging. The application of 3D printing to produce models of negative spaces, contrast CT radiographic data using segmentation software is illustrated. The accuracy of printed specimens is compared with original specimens. This alternative approach to producing anatomically accurate reproductions offers many advantages over plastination as it allows rapid production of multiple copies of any dissected specimen, at any size scale and should be suitable for any teaching facility in any country, thereby avoiding some of the cultural and ethical issues associated with cadaver specimens either in an embalmed or plastinated form., (© 2014 American Association of Anatomists.)

Aim: To evaluate vascularisation of the peripheral retina using fluorescein angiography (FA) digital recordings of infants who had been treated with intravitreal bevacizumab (IVB) as sole therapy for zone I and posterior zone II retinopathy of prematurity (ROP)., Methods: A retrospective evaluation was performed of medical records, RetCam fundus images and RetCam fluorescein angiogram videos of 10 neonates (20 eyes) who received intravitreal bevacizumab injections as the only treatment for zone I and posterior zone II ROP between August 2007 and November 2012., Results: All eyes had initial resolution of posterior disease after IVB injection as documented by RetCam colour fundus photographs. Using a distance of 2 disc diameters from the ora serrata to vascular termini as the upper limit of allowable avascular retina in children, the FA of these infants demonstrated that 11 of 20 eyes had not achieved normal retinal vascularisation., Conclusions: Although bevacizumab appears effective in bringing resolution of zone I and posterior zone II ROP and allowing growth of peripheral retinal vessels, in our series of 20 eyes, complete normal peripheral retinal vascularisation was not achieved in half of the patients.

Purpose: Immune and vascular alterations in the choroid are implicated in age-related macular degeneration (AMD). As choroidal immune cells are incompletely understood with regard to their physiology and interactions with choroidal vessels, we examined the associations between myeloid and vascular components of the choroid in young and aged mice., Methods: Albino CX3CR1(GFP/+) transgenic mice, whose choroidal myeloid cells possess green fluorescence, were perfused intraluminally with the vital dye DiI to label choroidal vessels. The distribution, morphology, behavior, and vascular associations of resident myeloid cells were examined using time-lapse live confocal imaging and immunohistochemical analysis., Results: Dendritiform myeloid cells, comprising most of the resident immune cell population in the choroid, were widely distributed across the choroid and demonstrated close associations with choroidal vessels that varied with their position in the vascular tree. Notably, myeloid cells associated with choroidal arteries and arterioles appeared as elongated cells flanking the long axes of vessels, whereas those associated with the choriocapillaris were distributed as a layer of stellate cells on the scleral but not vitreal choriocapillaris surface. While stationary in position, dendritiform myeloid cells demonstrated the rapid process dynamism well suited to comprehensive immunosurveillance of the perivascular space. Myeloid cells also increased in density as a function of aging, correlating locally with greater choroidal vascular attenuation., Conclusions: Resident myeloid cells demonstrated close but dynamic physical interactions with choroidal vessels, indicative of constitutive immune-vascular interactions in the normal choroid. These interactions may alter progressively with aging, providing a basis for understanding age-related choroidal dysfunction underlying AMD.

Purpose: To characterize the interactions between resident macrophage populations and nerves in naïve and injured corneas of the mouse eye., Methods: Corneas from wild-type (WT) C57BL/6J, BALB/cJ, and transgenic Cx3cr1-eGFP mice were subjected to a 1-mm central epithelial debridement injury. The eyes were fixed and immunostained as flat mounts with a range of antibodies to identify macrophages, neurons, and Schwann cells. Interactions between nerves and immune cells were analyzed and quantitated using three-dimensional reconstructions of confocal microscopy images. Naïve eyes acted as controls., Results: A distinctive association between resident immune cells and corneal nerves was noted in the peripheral or perilimbal stromal nerve trunks. These epineurial cells were mostly Cx3cr1(+) Iba-1(+) major histocompatibility complex (MHC) class II(+) F4/80(+) CD11b(+) macrophages. The number of nerve-associated macrophages was greater in WT BALB/c mice than in C57BL/6J mice. There were no qualitative or quantitative differences in the circumferential distribution of nerve-associated macrophages in the cornea. Sterile corneal epithelial debridement led to a dissociation of macrophages from peripheral nerve trunks as early as 2 hours postinjury, with numbers returning to baseline after 72 hours. This dissociation was Cx3cr1 dependent., Conclusions: This study is the first to highlight a direct physical association between nerves and resident immune cells in the murine cornea. Furthermore, we reveal that this association in normal eyes is responsive to central corneal epithelial injury and is partly mediated by Cx3cr1 signaling. This association may serve as an indicator of malfunctioning neuroimmune communication in disease states such as neurotrophic keratitis and peripheral neuropathy.

Aims: To evaluate the impact of systemic exposure to bacterial lipopolysaccharide (LPS) on a rodent model of background diabetic retinopathy., Methods: Toll-like receptor 4 (TLR4)-mediated systemic inflammation was induced in Ins2(Akita) heterozygotes and age-matched C57BL6/J-Ins2(+) littermates by single or repeated intraperitoneal injections of the TLR4 ligand LPS (9 µg/g body weight). 24 hours after a single injection in 7-week-old mice retinal Il1b, Tnfa and Vegf transcripts were measured with real-time PCR. Vascular endothelial growth factor (VEGF) protein levels were evaluated with bead-based immunoassay. Leukostasis and endothelial injury were assessed in retinal wholemounts following perfusion with rhodamine or FITC conjugated concanavalin A to label leukocytes and propidium iodide to label dead or injured cells. In mice which had received three fortnightly injections between 10 and 16 weeks of age, retinal thicknesses and vascular structure were evaluated at 17-18 weeks of age using optical coherence tomography (OCT) and fluorescein angiography. Retinal architecture was assesed using resin-based histology., Results: Compared with normoglycaemic controls, systemic LPS exposure in Ins2(Akita) mice was associated with a 3.5-fold increase in endothelial cell injury and attenuated leukostasis in the retinal vasculature. Hyperglycaemia or acute LPS inflammation did not increase retinal VEGF content. Thinning (10-13 µm) of posterior retina was detected with OCT 2 weeks after repeated exposure to LPS in Ins2(Akita) mice but not in normoglycaemic controls. Capillary networks and retinal morphology were unaffected by recurrent LPS inflammation in Ins2(Akita) and control mice., Conclusions: In hyperglycaemic mice, exposure to systemic LPS was associated with two hallmark pathologies of early background diabetic retinopathy, namely, the injury of capillary endothelium and in vivo thinning of the retina.

There has been considerable debate about whether dendritic cells (DCs), which are potent antigen-presenting cells pivotal to adaptive immune responses, are present in CNS parenchyma. In studies aimed at answering this issue, we discovered that while the neural retina of young naive transgenic C57BL/6 CD11c-eYFP reporter mice contained more than 800 CD11c-positive cells/retina, these cells were virtually absent in C57BL/6 CD11c-DTR/GFP mice. Clinical fundus examination, confocal imaging of retinal whole mounts, and sections revealed colocalization of CD11c-positive cells with classic mild to severe retinal dystrophic lesions. Immunophenotypic analysis revealed that CD11c-positive cells in the neural retina of these mice had the characteristic profile of activated microglia and not DCs. Genotypic analysis confirmed that the cause of the retinal dystrophic lesions in CD11c-eYFP transgenic mice was the occurrence of the Crb1(rd8) mutation, which affects all mice of the C57BL/6N strain but not the C57BL/6J strain. Comparison of 2 different types of CD11c reporter transgenic mice revealed that a mutation in the Crb1 gene leads to retinal degeneration resulting in the activation of large numbers of local microglia that could be readily mistaken for CD11c-positive putative DCs.

Background: The Ins2(Akita) mouse has been reported to display retinal pathology degeneration associated with advanced diabetic retinopathy. In the present study, we monitored retinal changes in these mice to establish if this model displays clinical features associated with advanced diabetic retinopathy in human patients., Methods: Ins2(Akita) mice (n = 55) on a C57Bl/6J background were monitored clinically from 9 to 25 weeks of age using a combination of scanning laser ophthalmoscopy, fluorescein angiography and optical coherence tomography. After clinical imaging, eyes were processed for immunostaining to examine microglial, astroglial and Muller glial responses to hyperglycaemia. To complement our optical coherence tomography imaging, retinal morphology and thicknesses were examined in high-quality semi-thin sections., Results: No retinal thinning or disruption of retinal architecture was observed by optical coherence tomography or resin histology in Ins2(Akita) mice up to 6 months of age. In addition, no vascular changes were detected by fluorescein angiography or by scanning laser ophthalmoscopy. With the exception of microglial activation, reduced glial fibrillary acid protein expression in astrocytes and an increase in glial fibrillary acid protein expression by Muller cells, no other changes were observed in the Ins2(Akita) retina., Conclusions: Our results indicate that the classical clinical correlates of human diabetic retinopathy are absent in Ins2(Akita) mice up to 6 months of age suggesting that either the histopathological processes underlying the development of diabetic retinopathy in this model require longer than 5 months of hyperglycaemia to result in disruption of retinal architecture or that advanced diabetic retinopathy is not a feature of the Ins2(Akita) diabetic mouse., (© 2013 The Authors. Clinical and Experimental Ophthalmology © 2013 Royal Australian and New Zealand College of Ophthalmologists.)