Impact of Obesity and Age on Mouse Corneal Innervation at the Epithelial-Stromal Interface.

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.

ß2-glycoprotein I promotes the clearance of circulating mitochondria.

ß2-glycoprotein I (ß2-Gp1) is a cardiolipin-binding plasma glycoprotein. It is evolutionarily conserved from invertebrates, and cardiolipin-bound ß2-Gp1 is a major target of antiphospholipid antibodies seen in autoimmune disorders. Cardiolipin is almost exclusively present in mitochondria, and mitochondria are present in circulating blood. We show that ß2-Gp1 binds to cell-free mitochondria (CFM) in the circulation and promotes its phagocytosis by macrophages at physiological plasma concentrations. Exogenous CFM had a short circulation time of less than 10 minutes in mice. Following infusion of CFM, ß2-Gp1-deficient mice had significantly higher levels of transfused mitochondria at 5 minutes (9.9 ± 6.4 pg/ml versus 4.0 ± 2.3 pg/ml in wildtype, p = 0.01) and at 10 minutes (3.0 ± 3.6 pg/ml versus 1.0 ± 0.06 pg/ml in wild-type, p = 0.033, n = 10). In addition, the splenic macrophages had less phagocytosed CFM in ß2-Gp1-deficient mice (24.4 ± 2.72% versus 35.6 ± 3.5 in wild-type, p = 0.001, n = 5). A patient with abnormal ß2-Gp1, unable to bind cardiolipin, has increased CFM in blood (5.09 pg/ml versus 1.26 ± 1.35 in normal) and his plasma induced less phagocytosis of CFM by macrophages (47.3 ± 1.6% versus 54.3 ± 1.3, p = 0.01) compared to normal plasma. These results show the evolutionarily conserved ß2-Gp1 is one of the mediators of the clearance of CFM in circulation.

Opposing Roles for the α Isoform of the Catalytic Subunit of Protein Phosphatase 1 in Inside-Out and Outside-In Integrin Signaling in Murine Platelets.

Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside-out and integrin αIIbß3-initiated outside-in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα-/- mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbß3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα-/- platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα-/- platelets and the p38 inhibitor blocked the increased integrin-mediated outside-in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα-/- mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.

A Low-Fat/Sucrose Diet Rich in Complex Carbohydrates Reverses High-Fat/Sucrose Diet-Induced Corneal Dysregulation.

High-fat/sucrose diet feeding in mice causes loss of corneal nerve function and impairs corneal wound healing. While changing to a diet with a low fat/sugar composition and enrichments in complex carbohydrates mitigates the reduction in nerve function, it remains to be determined if it has an effect on corneal wound healing. In this study, 6-week-old C57BL/6 male mice were fed either a normal diet or a high-fat/sucrose diet for 20 weeks. A third group (diet reversal) was placed on a high-fat/sucrose diet for 10 weeks followed by a normal diet for an additional 10 weeks. A central corneal epithelial abrasion wound was created, and wound closure was monitored. Neutrophil and platelet recruitment was assessed by immunofluorescence microscopy. Mice fed the high-fat/sucrose diet-only had greater adiposity (p < 0.005) than normal diet-only fed mice; diet reversal markedly reduced adiposity. Following corneal abrasion, wound closure was delayed by ~6 h (p ≤ 0.01) and, at 30 h post-wounding, fewer neutrophils reached the wound center and fewer extravascular platelets were present at the limbus (p < 0.05). Diet restored normal wound closure and neutrophil and platelet influx in the injured cornea. These data suggest compositional changes to the diet may be an effective diet-based therapeutic strategy for maintaining or restoring corneal health.

A novel interaction between extracellular vimentin and fibrinogen in fibrin formation.

INTRODUCTION: Thrombosis is frequently manifested in critically ill patients with systemic inflammation, including sepsis and COVID-19. The coagulopathy in systemic inflammation is often associated with increased levels of fibrinogen and D-dimer. Because elevated levels of vimentin have been detected in sepsis, we sought to investigate the relationship between vimentin and the increased fibrin formation potential observed in these patients. MATERIALS AND METHODS: This hypothesis was examined by using recombinant human vimentin, anti-vimentin antibodies, plasma derived from healthy and critically ill patients, confocal microscopy, co-immunoprecipitation assays, and size exclusion chromatography. RESULTS: The level of vimentin in plasma derived from critically ill subjects with systemic inflammation was on average two-fold higher than that of healthy volunteers. We determined that vimentin directly interacts with fibrinogen and enhances fibrin formation. Anti-vimentin antibody effectively blocked fibrin formation ex vivo and caused changes in the fibrin structure in plasma. Additionally, confocal imaging demonstrated plasma vimentin enmeshed in the fibrin fibrils. Size exclusion chromatography column and co-immunoprecipitation assays demonstrated a direct interaction between extracellular vimentin and fibrinogen in plasma from critically ill patients but not in healthy plasma. CONCLUSIONS: The results describe that extracellular vimentin engages fibrinogen in fibrin formation. In addition, the data suggest that elevated levels of an apparent aberrant extracellular vimentin potentiate fibrin clot formation in critically ill patients with systemic inflammation; consistent with the notion that plasma vimentin contributes to the pathogenesis of thrombosis.

Histone-stimulated platelet adhesion to mouse cremaster venules in vivo is dependent on von Willebrand factor.

OBJECTIVE: Extracellular histones are known mediators of platelet activation, inflammation, and thrombosis. Von Willebrand Factor (vWF) and Toll-like receptor 4 (TLR4) have been implicated in pro-inflammatory and prothrombotic histone responses. The objective of this study was to assess the role of vWF and TLR4 on histone-induced platelet adhesion in vivo. METHODS: Intravital microscopy of the mouse cremaster microcirculation, in the presence of extracellular histones or saline control, was conducted in wild-type, vWF-deficient, and TLR4-deficient mice to assess histone-mediated platelet adhesion. Platelet counts following extracellular histone exposure were conducted. Platelets were isolated from vWF-deficient mice and littermates to assess the role of vWF on histone-induced platelet aggregation. RESULTS: Histones promoted platelet adhesion to cremaster venules in vivo in wild-type animals, as well as in TLR4-deficient mice to a comparable degree. Histones did not lead to increased platelet adhesion in vWF-deficient mice, in contrast to littermate controls. In all genotypes, histones resulted in thrombocytopenia. Histone-induced platelet aggregation ex vivo was similar in vWF-deficient mice and littermate controls. CONCLUSIONS: Histone-induced platelet adhesion to microvessels in vivo is vWF-dependent and TLR4-independent. Platelet-derived vWF was not necessary for histone-induced platelet aggregation ex vivo. These data are consistent with the notion that endothelial vWF, rather than platelet vWF, mediates histone-induced platelet adhesion in vivo.

High levels of von Willebrand factor with reduced specific activities in hospitalized patients with or without COVID-19.

The COVID-19 pandemic is often accompanied by severe respiratory illness and thrombotic complications. Von Willebrand Factor (VWF) levels are highly elevated in this condition. However, limited data are available on the qualitative activity of VWF in COVID-19. We measured plasma VWF levels quantitatively (VWF antigen) and qualitatively (ristocetin-induced platelet agglutination, glycoprotein IbM (GPIbM) binding, and collagen binding). Consistent with prior reports, VWF antigen levels were significantly elevated in hospitalized patients with or without COVID-19. The GPIbM and collagen binding activity-to-antigen ratios were significantly reduced, consistent with qualitative changes in VWF in COVID-19. Of note, critically ill hospitalized patients without COVID-19 had similar reductions in VWF activity-to-antigen ratios as patients with COVID-19. Our data suggest that qualitative changes in VWF in COVID-19 may not be specific to COVID-19. Future studies are warranted to determine the mechanisms responsible for qualitative changes in VWF in COVID-19 and other critical illnesses.• VWF levels were increased in COVID-19 compared to healthy controls.• VWF activity-to-antigen ratios were decreased in COVID-19 compared to healthy controls.• There were no differences in VWF activity-to-antigen ratios between hospitalized patients with or without COVID-19.• These findings are consistent with qualitative changes in VWF in systemic inflammation which are not specific to COVID-19.• Future studies are needed to define possible roles of changes in conformation or multimer length in the qualitative changes in VWF in systemic inflammation.

Platelet and Erythrocyte Extravasation across Inflamed Corneal Venules Depend on CD18, Neutrophils, and Mast Cell Degranulation.

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the ß2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples.

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Dissociation between Corneal and Cardiometabolic Changes in Response to a Time-Restricted Feeding of a High Fat Diet.

Mice fed a high fat diet (HFD) ab libitum show corneal dysregulation, as evidenced by decreased sensitivity and impaired wound healing. Time-restricted (TR) feeding can effectively mitigate the cardiometabolic effects of an HFD. To determine if TR feeding attenuates HFD-induced corneal dysregulation, this study evaluated 6-week-old C57BL/6 mice fed an ad libitum normal diet (ND), an ad libitum HFD, or a time-restricted (TR) HFD for 10 days. Corneal sensitivity was measured using a Cochet-Bonnet aesthesiometer. A corneal epithelial abrasion wound was created, and wound closure was monitored for 30 h. Neutrophil and platelet recruitment were assessed by immunofluorescence microscopy. TR HFD fed mice gained less weight (p < 0.0001), had less visceral fat (p = 0.015), and had reduced numbers of adipose tissue macrophages and T cells (p < 0.05) compared to ad libitum HFD fed mice. Corneal sensitivity was reduced in ad libitum HFD and TR HFD fed mice compared to ad libitum ND fed mice (p < 0.0001). Following epithelial abrasion, corneal wound closure was delayed (~6 h), and neutrophil and platelet recruitment was dysregulated similarly in ad libitum and TR HFD fed mice. TR HFD feeding appears to mitigate adipose tissue inflammation and adiposity, while the cornea remains sensitive to the pathologic effects of HFD feeding.

An Epithelial Abrasion Model for Studying Corneal Wound Healing.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Links between thrombosis and inflammation in traumatic brain injury.

Traumatic brain injury (TBI) continues to be a major healthcare problem and there is much to be explored regarding the secondary pathobiology to identify early predictive markers and new therapeutic targets. While documented changes in thrombosis and inflammation in major trauma have been well described, growing evidence suggests that isolated TBI also results in systemic alterations in these mechanisms. Here, we review recent experimental and clinical findings that demonstrate how blood-brain barrier dysfunction, systemic immune response, inflammation, platelet activation, and thrombosis contribute significantly to the pathogenesis of TBI. Despite advances in the links between thrombosis and inflammation, there is a lack of treatment options aimed at both processes and this could be crucial to treating vascular injury, local and systemic inflammation, and secondary ischemic events following TBI. With emerging evidence of newly-identified roles for platelets, leukocytes, the coagulation system and extracellular vesicles in processes of inflammation and thrombosis, there is a growing need to characterize these mechanisms within the context of TBI and whether these changes persist into the chronic phase of injury. Importantly, this review defines areas in need of further research to advance the field and presents a roadmap to identify new diagnostic and treatment options for TBI.

Obese Mice with Dyslipidemia Exhibit Meibomian Gland Hypertrophy and Alterations in Meibum Composition and Aqueous Tear Production.

BACKGROUND: Dyslipidemia may be linked to meibomian gland dysfunction (MGD) and altered meibum lipid composition. The purpose was to determine if plasma and meibum cholesteryl esters (CE), triglycerides (TG), ceramides (Cer) and sphingomyelins (SM) change in a mouse model of diet-induced obesity where mice develop dyslipidemia. METHODS: Male C57/BL6 mice (8/group, age = 6 wks) were fed a normal (ND; 15% kcal fat) or an obesogenic high-fat diet (HFD; 42% kcal fat) for 10 wks. Tear production was measured and meibography was performed. Body and epididymal adipose tissue (eAT) weights were determined. Nano-ESI-MS/MS and LC-ESI-MS/MS were used to detect CE, TG, Cer and SM species. Data were analyzed by principal component analysis, Pearson's correlation and unpaired t-tests adjusted for multiple comparisons; significance set at p ≤ 0.05. RESULTS: Compared to ND mice, HFD mice gained more weight and showed heavier eAT and dyslipidemia with higher levels of plasma CE, TG, Cer and SM. HFD mice had hypertrophic meibomian glands, increased levels of lipid species acylated by saturated fatty acids in plasma and meibum and excessive tear production. CONCLUSIONS: The majority of meibum lipid species with saturated fatty acids increased with HFD feeding with evidence of meibomian gland hypertrophy and excessive tearing. The dyslipidemia is associated with altered meibum composition, a key feature of MGD.

Corneal dysfunction precedes the onset of hyperglycemia in a mouse model of diet-induced obesity.

PURPOSE: The purpose of this study was to use a mouse model of diet-induced obesity to determine if corneal dysfunction begins prior to the onset of sustained hyperglycemia and if the dysfunction is ameliorated by diet reversal. METHODS: Six-week-old male C57BL/6 mice were fed a high fat diet (HFD) or a normal diet (ND) for 5-15 weeks. Diet reversal (DiR) mice were fed a HFD for 5 weeks, followed by a ND for 5 or 10 weeks. Corneal sensitivity was determined using aesthesiometry. Corneal cytokine expression was analyzed using a 32-plex Luminex assay. Excised corneas were prepared for immunofluorescence microscopy to evaluate diet-induced changes and wound healing. For wounding studies, mice were fed a HFD or a ND for 10 days prior to receiving a central 2mm corneal abrasion. RESULTS: After 10 days of HFD consumption, corneal sensitivity declined. By 10 weeks, expression of corneal inflammatory mediators increased and nerve density declined. While diet reversal restored nerve density and sensitivity, the corneas remained in a heightened inflammatory state. After 10 days on the HFD, corneal circadian rhythms (limbal neutrophil accumulation, epithelial cell division and Rev-erbα expression) were blunted. Similarly, leukocyte recruitment after wounding was dysregulated and accompanied by delays in wound closure and nerve recovery. CONCLUSION: In the mouse, obesogenic diet consumption results in corneal dysfunction that precedes the onset of sustained hyperglycemia. Diet reversal only partially ameliorated this dysfunction, suggesting a HFD diet may have a lasting negative impact on corneal health that is resistant to dietary therapeutic intervention.

Microvascular thrombosis: experimental and clinical implications.

A significant amount of clinical and research interest in thrombosis is focused on large vessels (eg, 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.

Modulating the rate of fibrin formation and clot structure attenuates microvascular thrombosis in systemic inflammation.

Systemic inflammation can lead to coagulopathy and disseminated intravascular coagulation (DIC). In prior studies, the recombinant A2 domain of human von Willebrand factor (VWF; A2 protein) attenuated DIC and decreased mortality in lipopolysaccharide (LPS)-treated mice. Here, we performed studies to dissect the mechanism by which the A2 protein moderates DIC. We used confocal microscopy to analyze the fibrin clot structure in plasma from healthy humans and endotoxemic mice, turbidity assays to examine fibrin polymerization, and a murine model for LPS-induced DIC and introduced a loss-of-function mutation into the A2 protein for fibrin. The mutation of the residue E1567 located in the α2 helix of the folded A2 domain of VWF inhibited binding activity for fibrin, possibly mapping a novel region containing a putative binding site for fibrin. The A2 protein increased the initial rate of change of fibrin polymerization, intercalated into the fibrin network, and modified the resultant clot structure in vitro. Furthermore, ex vivo experiments using plasma from mice with endotoxemia treated with the A2 protein revealed an increased rate of fibrin formation and an altered clot structure as compared with plasma from nontreated sick animals. Moreover, and in contrast to the A2 mutant, the A2 protein improved survival and reduced fibrin deposition and microvascular thrombosis in mice with endotoxemia-induced DIC. Importantly, in vivo and in vitro studies indicated that the A2 protein did not affect experimental thrombosis. Thus, we provide evidence for a novel treatment to attenuate systemic inflammation-induced coagulopathy/DIC via targeting fibrin formation, without an increased risk for bleeding.

Serial block-face scanning electron microscopy reveals neuronal-epithelial cell fusion in the mouse cornea.

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 epithelium in the cornea fused with basal epithelial cells, such that their plasma membranes were continuous and the neuronal axoplasm freely abutted the epithelial cytoplasm. In this study we sought to determine the frequency, distribution, and morphological profile of neuronal-epithelial cell fusion events within the cornea. Serial electron microscopy images were obtained from the anterior stroma in the paralimbus and central cornea of 8-10 week old C57BL/6J mice. We found evidence of a novel alternative behavior involving a neuronal-epithelial interaction whereby 42.8% of central corneal nerve bundles approaching the epithelium contain axons that fuse with basal epithelial cells. The average surface-to-volume ratio of a penetrating nerve was 3.32, while the average fusing nerve was smaller at 1.39 (p ≤ 0.0001). Despite this, both neuronal-epithelial cell interactions involve similarly sized discontinuities in the basal lamina. In order to verify the plasma membrane continuity between fused neurons and epithelial cells we used the lipophilic membrane tracer DiI. The majority of corneal nerves were labeled with DiI after application to the trigeminal ganglion and, consistent with our ultrastructural observations, fusion sites recognized as DiI-labeled basal epithelial cells were located at points of stromal nerve termination. These studies provide evidence that neuronal-epithelial cell fusion is a cell-cell interaction that occurs primarily in the central cornea, and fusing nerve bundles are morphologically distinct from penetrating nerve bundles. This is, to our knowledge, the first description of neuronal-epithelial cell fusion in the literature adding a new level of complexity to the current understanding of corneal innervation.

Dyslipidemia and Meibomian Gland Dysfunction: Utility of Lipidomics and Experimental Prospects with a Diet-Induced Obesity Mouse Model.

Meibomian gland dysfunction (MGD) is the leading cause of dry eye disease and loss of ocular surface homeostasis. Increasingly, several observational clinical studies suggest that dyslipidemia (elevated blood cholesterol, triglyceride or lipoprotein levels) can initiate the development of MGD. However, conclusive evidence is lacking, and an experimental approach using a suitable model is necessary to interrogate the relationship between dyslipidemia and MGD. This systematic review discusses current knowledge on the associations between dyslipidemia and MGD. We briefly introduce a diet-induced obesity model where mice develop dyslipidemia, which can serve as a potential tool for investigating the effects of dyslipidemia on the meibomian gland. Finally, the utility of lipidomics to examine the link between dyslipidemia and MGD is considered.

Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis.

Platelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis.