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.

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.

PI3K activation within ventromedial prefrontal cortex regulates the expression of drug-seeking in two rodent species.

Phosphatidylinositide 3-kinases (PI3Ks) are intracellular signal transducer enzymes that recruit protein kinase B (aka Akt) to the cell membrane, the subsequent activation of which regulates many cellular functions. PI3K/Akt activity is up-regulated within mesocorticolimbic structures in animal models of alcoholism, but less is known regarding PI3K/Akt activity in animal models of cocaine addiction. Given that prefrontal cortex (PFC) is grossly dysregulated in addiction, we studied how cocaine affects protein indices of PFC PI3K/Akt activity in rat and mouse models and examined the relevance of PI3K activity for cocaine-related learning. Immunoblotting of mouse medial PFC at 3 weeks withdrawal from a cocaine-sensitization regimen (seven injections of 30 mg/kg, intraperitoneal [IP]) revealed increased kinase activity, as did immunoblotting of tissue from the ventral PFC of rats with a history of long-access intravenous cocaine self-administration (0.25 mg/0.1 mL infusion; 10 days of 6 h/d cocaine access). Interestingly, increased Akt phosphorylation was observed in rat ventromedial PFC at both 3- and 30-day withdrawal only in animals re-exposed to cocaine-associated cues. A conditioned place-preference paradigm in mice and a cue-elicited drug-seeking test in rats were conducted to determine the functional relevance for elevated PI3K activity for addiction-related behavior. In both cases, an intra-PFC infusion of the PI3K inhibitor wortmannin (50µM) reduced drug-seeking behavior. Taken together, this cross-species, interdisciplinary, study provides convincing evidence that cocaine history produces an enduring increase in PI3K/Akt-dependent signaling within the more ventral aspect of the PFC that is relevant to behavioral reactivity to drug-associated cues/contexts. As such, PI3K inhibitors may well serve as an effective strategy for reducing drug cue reactivity and craving in cocaine addiction.

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.

The impact of von Willebrand factor on fibrin formation and structure unveiled with type 3 von Willebrand disease plasma.

Normally, von Willebrand factor (VWF) remains inactive unless its A1A2 domains undergo a shear stress-triggered conformational change. We demonstrated the capacity of a recombinant A2 domain of VWF to bind and to affect fibrin formation, altering the fibrin clot structure. The data indicated that VWF contains an additional binding site for fibrin in the A2 domain that plays a role in the incorporation of VWF to the polymerizing fibrin. This study is to examine the hypothesis that active plasma VWF directly influence fibrin polymerization and the structure of fibrin clots. The study used healthy and type 3 von Willebrand disease (VWD) plasma, purified plasma VWF, fibrin polymerization assays, confocal microscopy and scanning electron microscopy. The exposed A2 domain in active VWF harbors additional binding sites for fibrinogen, and significantly potentiates fibrin formation (P < 0.02). Antibody against the A2 domain of VWF significantly decreased the initial rate of change of fibrin formation (P < 0.002). Clot analyses revealed a significant difference in porosity between normal and type 3 VWD plasma (P < 0.008), further supported by scanning electron microscopy, which demonstrated thicker fibrin fibers in the presence of plasma VWF (P < 0.0003). Confocal immunofluorescence microscopy showed punctate VWF staining along fibrin fibrils, providing visual evidence of the integration of plasma VWF into the fibrin matrix. The study with type 3 VWD plasma supports the hypothesis that plasma VWF directly influences fibrin polymerization and clot structure. In addition, a conformational change in the A1A2 domains exposes a hidden fibrin(ogen) binding site, indicating that plasma VWF determines the fibrin clot structure.

Nucleus accumbens mGluR5-associated signaling regulates binge alcohol drinking under drinking-in-the-dark procedures.

BACKGROUND: Alcohol increases the expression of Group 1 metabotropic glutamate receptors (mGluRs) and their associated scaffolding protein Homer2 and stimulates phosphatidylinositol 3-kinase (PI3K) within the nucleus accumbens (NAC). Moreover, functional studies suggest that NAC Group 1 mGluR/Homer2/PI3K signaling may be a potential target for pharmacotherapeutic intervention in alcoholism. METHODS: Immunoblotting was conducted to examine the effects of alcohol consumption under drinking-in-the-dark (DID) procedures on Group 1 mGluR-associated proteins in C57BL/6J (B6) mice. Follow-up behavioral studies examined the importance of Group 1 mGluR/Homer2/PI3K signaling within the NAC shell for limited-access alcohol drinking. Finally, immunoblotting examined whether the NAC expression of Group 1 mGluR-associated proteins is a genetic correlate of high alcohol drinking using a selectively bred high DID (HDID-1) mouse line. RESULTS: Limited-access alcohol drinking under DID procedures up-regulated NAC shell Homer2 levels, concomitant with increases in mGluR5 and NR2B. Intra-NAC shell blockade of mGluR5, Homer2, or PI3K signaling, as well as transgenic disruption of the Homer binding site on mGluR5, decreased alcohol consumption in B6 mice. Moreover, transgenic disruption of the Homer binding site on mGluR5 and Homer2 deletion both prevented the attenuating effect of mGluR5 and PI3K blockade upon intake. Finally, the basal NAC shell protein expression of mGluR1 and Homer2 was increased in offspring of HDID-1 animals. CONCLUSIONS: Taken together, these data further implicate Group 1 mGluR signaling through Homer2 within the NAC in excessive alcohol consumption.

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver.

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

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.

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.

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.

MyD88 Deficiency Protects Against Dry Eye-Induced Damage.

Purpose: Dry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation. Toll-like receptors (TLRs) are integral in the initiation of inflammatory signaling. Therefore, we evaluated the effect of TLR-deficiency on dry eye-related ocular surface damage and inflammation using a mouse model of experimental dry eye (EDE). Methods: C57BL/6 wild-type (WT), MyD88-/-, and IL-1R-/- mice were exposed to EDE conditions for 5 days. Tear production was measured by phenol red thread test and ocular surface damage assessed with fluorescein staining. Corneal homogenates were obtained for matrix metalloproteinase (MMP) and cytokine expression analysis by Luminex assay and quantitative PCR. In addition, whole eyes and eyelids were dissected and goblet cells and Meibomian glands were imaged, respectively. Results: Following 5 days of EDE, WT mice had extensive ocular surface staining, while MyD88-/- mice had no increased staining above non-EDE conditions. Similarly, MyD88-/- mice did not have increased corneal MMP-2, 3, or 8 concentrations, as seen with WT mice. MyD88-deficiency also resulted in decreased corneal cytokine levels. In addition, MyD88-/- mice had significantly lower conjunctival goblet cell counts compared with both WT (EDE) and IL-1R-/- (non-EDE) mice. However, there was no difference in Meibomian gland morphology between WT, IL-1R-/-, and MyD88-/- mice. Conclusions: These studies demonstrate the importance of TLR signaling in dry eye development. Mice lacking TLR signaling, MyD88-/-, were protected from EDE-induced ocular surface damage and inflammatory mediator expression, warranting further investigation into TLR inhibition as a potential therapeutic for DED.

MyD88 contribution to ocular surface homeostasis.

The cornea must maintain homeostasis, enabling rapid response to injury and microbial insult, to protect the eye from insult and infection. Toll-like receptors (TLRs) are critical to this innate immune response through the recognition and response to pathogens. Myeloid differentiation primary response (MyD88) is a key signaling molecule necessary for Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R)-mediated immune defense and has been shown to be necessary for corneal defense during infection. Here, we examined the intrinsic role of TLR signaling in ocular surface tissues by determining baseline levels of inflammatory mediators, the response to mechanical stimuli, and corneal infection in MyD88-deficient mice (MyD88-/-). In addition, cytokine, chemokine, and matrix metalloproteinase (MMP) expression was determined in ocular surface cells exposed to a panel of TLR agonists. Compared to wild-type (WT) animals, MyD88-/- mice expressed lower MMP-9 levels in the cornea and conjunctiva. Corneal IL-1α, TNFα, and conjunctival IL-1α, IL-2, IL-6, and IL-9 levels were also significantly reduced. Additionally, CXCL1 and RANTES expression was lower in both MyD88-/- tissues compared to WT and IL-1R-/- mice. Interestingly, MyD88-/- mice had lower corneal sensitivities (1.01±0.31 gm/mm2) than both WT (0.59±0.16 gm/mm2) and IL-1R-/- (0.52±0.08 gm/mm2). Following Pseudomonas aeruginosa challenge, MyD88-/- mice had better clinical scores (0.5±0.0) compared to IL-1R-/- (1.5±0.6) and WT (2.3±0.3) animals, but had significantly more corneal bacterial isolates. However, no signs of infection were detected in inoculated uninjured corneas from either MyD88 or IL-1R-deficient mice. This work furthers our understanding of the importance of TLR signaling in corneal defense and immune homeostasis, showing that a lack of MyD88 may compromise the baseline innate response to insult.

Protein Kinase C Epsilon Activity in the Nucleus Accumbens and Central Nucleus of the Amygdala Mediates Binge Alcohol Consumption.

BACKGROUND: Protein kinase C epsilon (PKCε) is emerging as a potential target for the development of pharmacotherapies to treat alcohol use disorders, yet little is known regarding how a history of a highly prevalent form of drinking, binge alcohol intake, influences enzyme priming or the functional relevance of kinase activity for excessive alcohol intake. METHODS: Immunoblotting was employed on tissue from subregions of the nucleus accumbens (NAc) and the amygdala to examine both idiopathic and binge drinking-induced changes in constitutive PKCε priming. The functional relevance of PKCε translocation for binge drinking and determination of potential upstream signaling pathways involved were investigated using neuropharmacologic approaches within the context of two distinct binge drinking procedures, drinking in the dark and scheduled high alcohol consumption. RESULTS: Binge alcohol drinking elevated p(Ser729)-PKCε levels in both the NAc and the central nucleus of the amygdala (CeA). Moreover, immunoblotting studies of selectively bred and transgenic mouse lines revealed a positive correlation between the propensity to binge drink alcohol and constitutive p(Ser729)-PKCε levels in the NAc and CeA. Finally, neuropharmacologic inhibition of PKCε translocation within both regions reduced binge alcohol consumption in a manner requiring intact group 1 metabotropic glutamate receptors, Homer2, phospholipase C, and/or phosphotidylinositide-3 kinase function. CONCLUSIONS: Taken together, these data indicate that PKCε signaling in both the NAc and CeA is a major contributor to binge alcohol drinking and to the genetic propensity to consume excessive amounts of alcohol.

Binge alcohol drinking by mice requires intact group 1 metabotropic glutamate receptor signaling within the central nucleus of the amygdala.

Despite the fact that binge alcohol drinking (intake resulting in blood alcohol concentrations (BACs) 80 mg% within a 2-h period) is the most prevalent form of alcohol-use disorders (AUD), a large knowledge gap exists regarding how this form of AUD influences neural circuits mediating alcohol reinforcement. The present study employed integrative approaches to examine the functional relevance of binge drinking-induced changes in glutamate receptors, their associated scaffolding proteins and certain signaling molecules within the central nucleus of the amygdala (CeA). A 30-day history of binge alcohol drinking (for example, 4-5 g kg(-1) per 2 h(-1)) elevated CeA levels of mGluR1, GluN2B, Homer2a/b and phospholipase C (PLC) ß3, without significantly altering protein expression within the adjacent basolateral amygdala. An intra-CeA infusion of mGluR1, mGluR5 and PLC inhibitors all dose-dependently reduced binge intake, without influencing sucrose drinking. The effects of co-infusing mGluR1 and PLC inhibitors were additive, whereas those of coinhibiting mGluR5 and PLC were not, indicating that the efficacy of mGluR1 blockade to lower binge intake involves a pathway independent of PLC activation. The efficacy of mGluR1, mGluR5 and PLC inhibitors to reduce binge intake depended upon intact Homer2 expression as revealed through neuropharmacological studies of Homer2 null mutant mice. Collectively, these data indicate binge alcohol-induced increases in Group1 mGluR signaling within the CeA as a neuroadaptation maintaining excessive alcohol intake, which may contribute to the propensity to binge drink.