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Application of DiI to the trigeminal ganglia of the mouse. A dissected view of the mouse cranial cavity showing trigeminal ganglia ( � ) straddling the optic chiasm. The trigeminal branches are labeled as 1, 2 and 3 and correspond to the ophthalmic, maxillary, and mandibular branches. DiI crystal was applied to the severed ophthalmic branch. https://doi.org/10.1371/journal.pone.0224434.g002
Source publication
The cornea is the most highly innervated tissue in the body. It is generally accepted that corneal stromal nerves penetrate the epithelial basal lamina giving rise to intra-epithelial nerves. During the course of a study wherein we imaged corneal nerves in mice, we observed a novel neuronal-epithelial cell interaction whereby nerves approaching the...
Context in source publication
Context 1
... head was then placed in 2% paraformaldehyde overnight. The following day, the trigeminal ganglion was located [31], severed at the ophthalmic branch, and DiI crystals (1, 1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, ThermoFisher, Waltham, MA) were crushed into the ganglia using surgical tweezers (Fig 2). The region around the ganglia was dried using chem-wipes prior to DiI application, as DiI is a hydrophobic substance [32,33]. ...
Citations
... Instead of glial cells, the basal membrane infoldings of corneal basal epithelial cells provide support to ICBNs as do Schwann cells elsewhere (Stepp et al., 2017). Thus, there is extensive contact between ICBNs and epithelial cells, and even scattered focal fusion of cell membranes between corneal axons and basal epithelial cells has been reported in mice (Courson et al., 2019). Second, as ICBNs converge centripetally, they form a characteristic whorl pattern (Al-Aqaba et al., 2010;Patel and McGhee, 2005) that is centered inferior to the corneal apex (Patel and McGhee, 2008). ...
As the cornea is densely innervated, its nerves are integral not only to its structure but also to its pathophysiology. Corneal integrity depends on a protective tear film that is maintained by corneal sensation and the reflex arcs that control tearing and blinking. Furthermore, corneal nerves promote epithelial growth and local immunoregulation. Thus, corneal nerves constitute pillars of ocular surface homeostasis. Conversely, the abnormal tear film in dry eye favors corneal epithelial and nerve damage. The ensuing corneal nerve dysfunction contributes to dry eye progression, ocular pain and discomfort, and other neuropathic symptoms. Recent evidence from clinical studies and animal models highlight the significant but often overlooked neural dimension of dry eye pathophysiology. Herein, we review the anatomy and physiology of corneal nerves before exploring their role in the mechanisms of dry eye disease.
... Mouse lungs were processed for serial block-face scanning electron microscopy (SBF-SEM) as described previously in detail [57]. Briefly, excised lungs were inflated through the trachea with fixative (0.1M sodium cacodylate buffer containing 2.5% glutaraldehyde), post-stained with heavy metals (Fe, OsO4, uranyl acetate, lead) before dehydration through an acetone series and embedding in Embed 812 resin (Embed-812, Electron Microscopy Sciences, Hatsfield, PA) containing Ketjen Black (Ketjenblack EC600JD, Lion Specialty Chemicals Co., Tokyo). ...
Ascariasis is one of the most common infections in the world and associated with significant global morbidity. Ascaris larval migration through the host’s lungs is essential for larval development but leads to an exaggerated type-2 host immune response manifesting clinically as acute allergic airway disease. However, whether Ascaris larval migration can subsequently lead to chronic lung diseases remains unknown. Here, we demonstrate that a single episode of Ascaris larval migration through lungs induces a chronic pulmonary syndrome of type-2 inflammatory pathology and emphysema accompanied by pulmonary hemorrhage and chronic anemia in a mouse model. Our results reveal that a single episode of Ascaris larval migration through the host lungs leads to permanent lung damage with systemic effects. Remote episodes of ascariasis may drive non-communicable lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), and chronic anemia in parasite endemic regions.
... Corneal epithelial nerve fibres travel primarily in infoldings of the basal cell basal plasmalemma [62,65]; this is also where most epithelial nerve endings are located. At intervals, the epithelial nerve fibres ascend between cells to the squamous cell layers, where they terminate at multiple levels within the corneal epithelium ( Fig. 2) [59,66]. ...
A key element of contact lens practice involves clinical evaluation of anterior eye health, including the cornea and limbus, conjunctiva and sclera, eyelids and eyelashes, lacrimal system and tear film. This report reviews the fundamental anatomy and physiology of these structures, including the vascular supply, venous drainage, lymphatic drainage, sensory innervation, physiology and function. This is the foundation for considering the potential interactions with, and effects of, contact lens wear on the anterior eye. This information is not consistently published as academic research and this report provides a synthesis from all available sources. With respect to terminology, the report aims to promote the consistent use of nomenclature in the field, and generally adopts anatomical terms recommended by the Federative Committee for Anatomical Terminology. Techniques for the examination of the ocular surface are also discussed.
... This timing aligns with observations of others [14,59] who reported the formation of epithelial and neural whorls at 4-weeks of age respectively. This phenomenon is not surprising, considering the close spatial and physical interactions these two cell-types share, whereby ICBNs are wrapped in epithelial cell membranes [62] or fuse [63] with epithelia which potentially 'tow' axons across the cornea as part of their life-long replenishing activity [3,14]. It is likely that physical and chemical cues function in tandem to mediate the interplay between these two cell types. ...
... These perforation sites have been documented in humans [9,60], however comprehensive surveys in animals are not available. A recent study in mouse described their ultrastructural morphology and noted that stromal axons which pass through these sites either fuse with basal corneal epithelia or contribute to the formation of the ICBN plexus [63]. Herein we report that the number and distribution of CSENPS remained relatively constant during post-natal development (Fig. 7A-F). ...
Purpose
How sensory neurons and epithelial cells interact with one another, and whether this association can be considered an indicator of health or disease is yet to be elucidated.
Methods
Herein, we used the cornea, Confetti mice, a novel image segmentation algorithm for intraepithelial corneal nerves which was compared to and validated against several other analytical platforms, and three mouse models to delineate this paradigm. For aging, eyes were collected from 2 to 52 week-old normal C57BL/6 mice (n ≥ 4/time-point). For wound-healing and limbal stem cell deficiency, 7 week-old mice received a limbal-sparing or limbal-to-limbal epithelial debridement to their right cornea, respectively. Eyes were collected 2–16 weeks post-injury (n=4/group/time-point), corneas procured, immunolabelled with βIII-tubulin, flat-mounted, imaged by scanning confocal microscopy and analysed for nerve and epithelial-specific parameters.
Results
Our data indicate that nerve features are dynamic during aging and their curvilinear arrangement align with corneal epithelial migratory tracks. Moderate corneal injury prompted axonal regeneration and recovery of nerve fiber features. Limbal stem cell deficient corneas displayed abnormal nerve morphology, and fibers no longer aligned with corneal epithelial migratory tracks. Mechanistically, we discovered that nerve pattern restoration relies on the number and distribution of stromal-epithelial nerve penetration sites.
Conclusions
Microstructural changes to innervation may explain corneal complications related to aging and/or disease and facilitate development of new assays for diagnosis and/or classification of ocular and systemic diseases.
... Fig 3 demonstrates the 3dimensional ultrastructure of a microvascular thrombus generated by our group using the photochemical injury model in a mouse cremaster venule (procedures approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine), using the relatively new imaging technique of serial block-face scanning electron microscopy. 116 Many of the experimental models of microvascular thrombosis described above result in platelet-rich thrombi, often with little evidence of fibrin by electron microscopy. 78 As described below, the structure of thrombi in clinical conditions associated with microvascular thrombosis varies considerably; for example, platelet-rich thrombi predominate in thrombotic thrombocytopenic purpura and fibrin-rich thrombi in hemolytic-uremic syndrome and DIC. ...
A significant amount of clinical and research interest in thrombosis is focused on large vessels (e.g., stroke, myocardial infarction, deep venous thrombosis, etc.); however, thrombosis is often present in the microcirculation in a variety of significant human diseases, such as disseminated intravascular coagulation, thrombotic microangiopathy, sickle cell disease, and others. Further, microvascular thrombosis has recently been demonstrated in patients with COVID-19, and has been proposed to mediate the pathogenesis of organ injury in this disease. In many of these conditions, microvascular thrombosis is accompanied by inflammation, an association referred to as thromboinflammation. In this review, we discuss endogenous regulatory mechanisms that prevent thrombosis in the microcirculation, experimental approaches to induce microvascular thrombi, and clinical conditions associated with microvascular thrombosis. A greater understanding of the links between inflammation and thrombosis in the microcirculation is anticipated to provide optimal therapeutic targets for patients with diseases accompanied by microvascular thrombosis.
... Our movies were acquired to focus on the epithelium. Courson and colleagues (Courson et al., 2019) have assessed sites where stromal nerves penetrate the EBM in adult mice using serial block face scanning electron microscopy (SBF-SEM). SBF-SEM allows larger fields of view (Peddie and Collinson, 2014); while that advantage comes at the cost of lower resolution, both SBF-SEM and FIB-SEM provide exceptional advantages compared to traditional TEM. ...
The intraepithelial corneal nerves (ICNs) that innervate the corneal epithelium are maintained through interactions with corneal epithelial cells and the extracellular matrix they produce. One to several axons bundle together within the basal cell layer and extend parallel to the ocular surface or branch and extend apically. Here we use 3-dimentional (3D) ultrastructural reconstructions of control and trephine injured mouse corneal epithelium and stroma produced using Focused Ion Beam Scanning Electron Microscope (FIB-SEM) to determine whether corneal epithelial or immune cells resident in the epithelium remove axonal debris and degrade it in their lysosomes after trephine injury to the cornea. We demonstrate that axonal fragments are internalized in the corneal epithelium and accumulate within electron dense structures consistent with lysosomes 3 h after trephine injury in both epithelial and immune cells located among the basal cells of the trephine injured cornea. Confocal imaging showed fewer CD45⁺ immune cells within the corneal epithelium after trephine injury compared to controls. The resolution obtained using FIB-SEM also allowed us to show that the presence of sensory axons at the basal aspect of the epithelial basal cells close to the anterior aspect of the epithelial basement membrane (EBM) is associated with a focal reduction in EBM thickness. In addition, we show using FIB-SEM and confocal imaging that superficial trephine injuries that do not penetrate the stroma, damage the integrity of anterior stromal nerves. These studies are the first to look at the mouse cornea following nerve injury using FIB-SEM.
Purpose
The corneal epithelium is the most highly innervated structure in the body. Previously, we reported a novel event whereby stromal axons fuse with basal epithelial cells, limiting nerve penetration into the epithelium. Although corneal–epithelial nerves undergo changes in sensitivity and distribution throughout life and in response to an obesogenic diet, it is unknown if neuronal–epithelial cell fusion is altered. Here, we sought to determine if neuronal–epithelial cell fusion frequency correlates with obesogenic diet consumption and age.
Methods
Corneas were collected from C57BL/6 mice and evaluated for neuronal–epithelial cell fusion frequency using serial block-face scanning electron microscopy. To assess the correlation between diet-induced obesity and fusion frequency, 6-week-old mice were fed either a normal diet or an obesogenic diet for 10 weeks. To assess changes in fusion frequency between young and adult mice under normal dietary conditions, 9- and 24-week-old mice were used.
Results
Mice fed a 10-week obesogenic diet showed 87% of central-cornea stromal nerves engaged in fusion compared with only 54% in age-matched controls (16 weeks old). In 9-week-old normal-diet animals, 48% of central-cornea stromal nerves contained fusing axons and increased to 81% at 24 weeks of age. Corneal sensitivity loss correlated with increased body weight and adiposity regardless of age and diet.
Conclusions
Neuronal–epithelial cell fusion positively correlates with age and obesogenic diet consumption, and corneal nerve sensitivity loss correlates with increased body weight and adiposity, regardless of age and diet. As such, neuronal–epithelial cell fusion may play a role in corneal nerve density and sensitivity regulation.
In the cornea, resident immune cells are in close proximity to sensory nerves, consistent with their important roles in the maintenance of nerves in both homeostasis and inflammation. Using in vivo confocal microscopy in humans, and ex vivo immunostaining and fluorescent reporter mice to visualize corneal sensory nerves and immune cells, remarkable progress has been made to advance our understanding of the physical and functional interactions between corneal nerves and immune cells. In this review, we summarize and discuss recent studies relating to corneal immune cells and sensory nerves, and their interactions in health and disease. In particular, we consider how disrupted corneal nerve axons can induce immune cell activity, including in dendritic cells, macrophages and other infiltrating cells, directly and/or indirectly by releasing neuropeptides such as substance P and calcitonin gene-related peptide. We summarize growing evidence that the role of corneal intraepithelial immune cells is likely different in corneal wound healing versus other inflammatory-dominated conditions. The role of different types of macrophages is also discussed, including how stromal macrophages with anti-inflammatory phenotypes communicate with corneal nerves to provide neuroprotection, while macrophages with pro-inflammatory phenotypes, along with other infiltrating cells including neutrophils and CD4⁺ T cells, can be inhibitory to corneal re-innervation. Finally, this review considers the bidirectional interactions between corneal immune cells and corneal nerves, and how leveraging this interaction could represent a potential therapeutic approach for corneal neuropathy.
Les épithéliums de la surface oculaire sont en première ligne pour protéger l'œil des agressions extérieures, notamment des environnements aujourd’hui de plus en plus pollués et toxiques responsables de l’augmentation de l’incidence de la sécheresse oculaire. La cornée et la conjonctive, innervées par les neurones du ganglion trigéminé vont répondre à ces agressions par la mise en place de processus inflammatoires et nociceptifs. Parmi les polluants auxquels nous sommes le plus exposés se trouvent le formaldéhyde gazeux (FA) et le chlorure de benzalkonium (BAK). Nous avons testé in vitro les effets toxiques de ces deux xénobiotiques sur des cellules épithéliales cornéennes et conjonctivales et sur des neurones trigéminés. Dans une première étude, nous avons cultivé en interface air-liquide, des cellules épithéliales conjonctivales humaines de la lignée WKD afin de pouvoir les exposer à un flux de FA modélisant un stress toxique. Dans une deuxième étude, nous avons évalué les interactions entre cellules épithéliales et neurones trigéminés suite à un stress toxique. Ainsi, une culture primaire de neurones trigéminés a été exposée à un milieu conditionné (CM) produit par des cellules épithéliales cornéennes de la lignée HCE préalablement exposées à du BAK. Dans une troisième étude, nous avons développé un modèle de compartimentalisation en microfluidique des neurones trigéminés, afin d'étudier les réponses neuronales lors d’un stress toxique au BAK appliqué au niveau des terminaisons axonales. Ainsi, l’ensemble de nos résultats met en exergue des mécanismes cellulaires et moléculaires mis en jeu lors d'un stress toxique sur la surface oculaire.
