Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro†.

Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus-oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.

Characterization of Resident Corneal Plasmacytoid Dendritic Cells and Their Pivotal Role in Herpes Simplex Keratitis.

The presence and potential functions of resident plasmacytoid dendritic cells (pDCs) in peripheral tissues is unclear. We report that pDCs constitutively populate naïve corneas and are increased during sterile injuries or acute herpes simplex virus 1 (HSV-1) keratitis. Their local depletion leads to severe clinical disease, nerve loss, viral dissemination to the trigeminal ganglion and draining lymph nodes, and mortality, while their local adoptive transfer limits disease. pDCs are the main source of HSV-1-induced IFN-α in the corneal stroma through TLR9, and they prevent re-programming of regulatory T cells (Tregs) to effector ex-Tregs. Clinical signs of infection are observed in pDC-depleted corneas, but not in pDC-sufficient corneas, following low-dose HSV-1 inoculation, suggesting their critical role in corneal antiviral immunity. Our findings demonstrate a vital role for corneal pDCs in the control of local viral infections.

Bilateral nerve alterations in a unilateral experimental neurotrophic keratopathy model: a lateral conjunctival approach for trigeminal axotomy.

To study bilateral nerve changes in a newly developed novel mouse model for neurotrophic keratopathy by approaching the trigeminal nerve from the lateral fornix. Surgical axotomy of the ciliary nerve of the trigeminal nerve was performed in adult BALB/c mice at the posterior sclera. Axotomized, contralateral, and sham-treated corneas were excised on post-operative days 1, 3, 5, 7 and 14 and immunofluorescence histochemistry was performed with anti-ß-tubulin antibody to evaluate corneal nerve density. Blink reflex was evaluated using a nylon thread. The survival rate was 100% with minimal bleeding during axotomy and a surgical time of 8±0.5 minutes. The blink reflex was diminished at day 1 after axotomy, but remained intact in the contralateral eyes in all mice. The central and peripheral subbasal nerves were not detectable in the axotomized cornea at day 1 (p<0.001), compared to normal eyes (101.3±14.8 and 69.7±12.0 mm/mm² centrally and peripherally). Interestingly, the subbasal nerve density in the contralateral non-surgical eyes also decreased significantly to 62.4±2.8 mm/mm² in the center from day 1 (p<0.001), but did not change in the periphery (77.3±11.7 mm/mm², P = 0.819). Our novel trigeminal axotomy mouse model is highly effective, less invasive, rapid, and has a high survival rate, demonstrating immediate loss of subbasal nerves in axotomized eyes and decreased subbasal nerves in contralateral eyes after unilateral axotomy. This model will allow investigating the effects of corneal nerve damage and serves as a new model for neurotrophic keratopathy.

Inflammation-induced intussusceptive angiogenesis in murine colitis.

Intussusceptive angiogenesis is a morphogenetic process that forms new blood vessels by the division of a single blood vessel into two lumens. Here, we show that this process of intraluminal division participates in the inflammation-induced neovascularization associated with chemically induced murine colitis. In studies of both acute (4-7 days) and chronic (28-31 days) colitis, intravital microscopy of intravascular tracers demonstrated a twofold reduction in blood flow velocity. In the acute colitis model, the decreased velocity was associated with marked dilatation of the mucosal plexus. In contrast, chronic inflammation was associated with normal caliber vessels and duplication (and triplication) of the quasi-polygonal mucosal plexus. Scanning electron microscopy (SEM) of intravascular corrosion casts suggested that pillar formation and septation, previously linked to the morphogenetic process of intussusceptive angiogenesis, were present within days of the onset of inflammation. Four weeks after the onset of inflammation, SEM of vascular corrosion casts demonstrated replication of the mucosal plexus without significant evidence of sprouting angiogenesis. These data suggest that mucosal capillaries have comparable aggregate cross-sectional area in acute and chronic colitis; however, there is a significant increase in functional capillary density in chronic colitis. We conclude that intussusceptive angiogenesis is a fundamental mechanism of microvascular adaptation to prolonged inflammation.

Blood flow patterns spatially associated with platelet aggregates in murine colitis.

In the normal murine mucosal plexus, blood flow is generally smooth and continuous. In inflammatory conditions, such as chemically-induced murine colitis, the mucosal plexus demonstrates markedly abnormal flow patterns. The inflamed mucosal plexus is associated with widely variable blood flow velocity as well as discontinuous and even bidirectional flow. To investigate the mechanisms responsible for these blood flow patterns, we used intravital microscopic examination of blood flow within the murine mucosal plexus during dextran sodium sulphate-and trinitrobenzenesulfonic acid-induced colitis. The blood flow patterns within the mucosal plexus demonstrated flow exclusion in 18% of the vessel segments (P < 0.01). Associated with these segmental exclusions was significant variation in neighboring flow velocities. Intravascular injection of fluorescent platelets demonstrated platelet incorporation into both fixed and rolling platelet aggregates. Rolling platelet aggregates (mean velocity 113 microm/sec; range, 14-186 microm/sec) were associated with reversible occlusions and flow variations within the mucosal plexus. Gene expression profiles of microdissected mucosal plexus demonstrated enhanced expression of genes for CCL3, CXCL1, CCL2, CXCL5, CCL7, CCL8, and Il-1b (P < 0.01), and decreased expression of CCL6 (P < 0.01). These results suggest that platelet aggregation, activated by the inflammatory mileau, contributes to the complex flow dynamics observed in acute murine colitis.

Vascular microarchitecture of murine colitis-associated lymphoid angiogenesis.

In permissive tissues, such as the gut and synovium, chronic inflammation can result in the ectopic development of anatomic structures that resemble lymph nodes. These inflammation-induced structures, termed lymphoid neogenesis or tertiary lymphoid organs, may reflect differential stromal responsiveness to the process of lymphoid neogenesis. To investigate the structural reorganization of the microcirculation involved in colonic lymphoid neogenesis, we studied a murine model of dextran sodium sulfate (DSS)-induced colitis. Standard 2-dimensional histology demonstrated both submucosal and intramucosal lymphoid structures in DSS-induced colitis. A spatial frequency analysis of serial histologic sections suggested that most intramucosal lymphoid aggregates developed de novo. Intravital microscopy of intravascular tracers confirmed that the developing intramucosal aggregates were supplied by capillaries arising from the quasi-polygonal mucosal plexus. Confocal optical sections and whole mount morphometry demonstrated capillary networks (185 +/- 46 microm diameter) involving six to ten capillaries with a luminal diameter of 6.8 +/- 1.1 microm. Microdissection and angiogenesis PCR array analysis demonstrated enhanced expression of multiple angiogenic genes including CCL2, CXCL2, CXCL5, Il-1b, MMP9, and TNF within the mucosal plexus. Intravital microscopy of tracer particle flow velocities demonstrated a marked decrease in flow velocity from 808 +/- 901 microm/sec within the feeding mucosal plexus to 491 +/- 155 microm/sec within the capillary structures. We conclude that the development of ectopic lymphoid tissue requires significant structural remodeling of the stromal microcirculation. A feature of permissive tissues may be the capacity for lymphoid angiogenesis.

Spatial calibration of structured illumination fluorescence microscopy using capillary tissue phantoms.

Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out-of-focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane-of-focus within the specimen. Because structured illumination can be applied to wide-field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell-sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral (X-Y) plane, but demonstrated a loss of resolution in the Z-Y plane. Because the magnitude of Z-axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z-axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation.

Bimodal oscillation frequencies of blood flow in the inflammatory colon microcirculation.

Rhythmic changes in blood flow direction have been described in the mucosal plexus of mice with acute colitis. In this report, we studied mice with acute colitis induced either by dextran sodium sulfate or by trinitrobenzenesulfonic acid. Both forms of colitis were associated with blood flow oscillations as documented by fluorescence intravital videomicroscopy. The complex oscillation patterns suggested more than one mechanism for these changes in blood flow. By tracking fluorescent nanoparticles in the inflamed mucosal plexus, we identified two forms of blood flow oscillations within the inflammatory mouse colon. Stable oscillations were associated with a base frequency of approximately 2 cycles/sec. Velocity measurements in the upstream and downstream vessel segments indicated that stable oscillations were the result of regional flow occlusion within the mucosal plexus. In contrast, metastable oscillations demonstrated a lower frequency (0.2-0.4 cycles/sec) and appeared to be the result of flow dynamics in vessels linked by the bridging mucosal vessels. These blood flow oscillations were not directly associated with cardiopulmonary movement. We conclude that both the stable and metasable oscillating patterns reflect flow adaptations to inflammatory changes in the mucosal plexus. Anat Rec, 2009. (c) 2008 Wiley-Liss, Inc.

Effect of intraluminal pillars on particle motion in bifurcated microchannels.

A central feature of intussusceptive angiogenesis is the development of an intravascular pillar that bridges the opposing sides of the microvessel lumen. In this report, we created polydimethyl siloxane (PDMS) microchannels with geometric proportions based on corrosion casts of the colon microcirculation. The structure of the PDMS microchannels was a bifurcated channel with an intraluminal pillar in the geometric center of the bifurcation. The effect of the intraluminal pillar on particle flow paths was investigated using an in vitro perfusion system. The microchannels were perfused with fluorescent particles, and the particle movements were recorded using fluorescence videomicroscopy. We found that the presence of an intravascular pillar significantly decreased particle velocity in the bifurcation system (p < 0.05). In addition, the pillar altered the trajectory of particles in the center line of the flow stream. The particle trajectory resulted in prolonged pillar contact as well as increased residence time within the bifurcation system (p < 0.001). Our results suggest that the intravascular pillar not only provides a mechanism of increasing resistance to blood flow but may also participate in spatial redistribution of cells within the flow stream.

Bridging mucosal vessels associated with rhythmically oscillating blood flow in murine colitis.

Oscillatory blood flow in the microcirculation is generally considered to be the result of cardiopulmonary influences or active vasomotion. In this report, we describe rhythmically oscillating blood flow in the bridging vessels of the mouse colon that appeared to be independent of known biological control mechanisms. Corrosion casting and scanning electron microscopy of the mouse colon demonstrated highly branched bridging vessels that connected the submucosal vessels with the mucosal plexus. Because of similar morphometric characteristics (19 +/- 11 microm vs. 28 +/- 16 microm), bridging arterioles and venules were distinguished by tracking fluorescent nanoparticles through the microcirculation using intravital fluorescence videomicroscopy. In control mice, the blood flow through the bridging vessels was typically continuous and unidirectional. In contrast, two models of chemically induced inflammation (trinitrobenzenesulfonic acid and dextran sodium sulfate) were associated with a twofold reduction in flow velocity and the prominence of rhythmically oscillating blood flow. The blood oscillation was characterized by tracking the bidirectional displacement of fluorescent nanoparticles. Space-time plots and particle tracking of the oscillating segments demonstrated an oscillation frequency between 0.2 and 5.1 cycles per second. Discrete Fourier transforms demonstrated a power spectrum composed of several base frequencies. These observations suggest that inflammation-inducible changes in blood flow patterns in the murine colon resulted in both reduced blood flow velocity and rhythmic oscillations within the bridging vessels of the mouse colon.

Biological and optical properties of fluorescent nanoparticles developed for intravascular imaging.

Intravascular tracers in the blood circulation can provide a description of the flow field over time and space. To address the limitations of existing intravascular tracers, we have developed fluorescent nanoparticles capable of providing detailed information regarding the intravascular flow field. The nanoparticles were designed to maximize plasma half-life as well as minimize interactions with other blood components. The bioavailability of the particles in the blood circulation required nanoscale size and low surface charge density. Intravital imaging of nanoparticles in the microcirculation demonstrated that the fluorescence intensity of the nanoparticles was a major determinant of both temporal and spatial resolution of the flow field. We conclude that nanoparticles prepared with these physical and optical properties can provide an accurate description of the localized intravascular flow field.

Adherent leukocytes capture sickle erythrocytes in an in vitro flow model of vaso-occlusion.

Recent in vivo studies suggest that adherent leukocytes bind RBCs and contribute to the microvascular pathology that characterizes sickle cell disease (SCD). A parallel-plate flow assay was used: to investigate the capture of RBCs by adherent neutrophils, monocytes, and T-lymphocytes; to examine whether RBC capture is elevated in patients with SCD; and to determine whether hydroxyurea (HU) therapy affects these interactions. Four measures of cell-cell adhesion were used: adhesion of leukocytes to TNF-alpha-treated human umbilical vein endothelial cells (HUVECs), percent of adherent leukocytes that captured RBCs, number of RBCs captured per interacting leukocyte, and duration of RBC capture. Leukocyte subpopulations from sickle patients were more adherent to activated ECs and captured more RBCs per interacting leukocyte than the corresponding subpopulations from healthy controls. While HU did not affect leukocyte adhesion to activated ECs, it reduced the proportion of adherent leukocytes that captured RBCs, as well as the number of RBCs captured per neutrophil. T-lymphocytes demonstrated elevated adhesion in all measures, and may be the leukocyte subpopulation whose behavior is most altered in SCD. Our findings suggest that neutrophils, monocytes, and T-lymphocytes could all be involved in adhesive interactions with autologous RBCs in patients with SCD.

Multiframe particle tracking in intravital imaging: defining Lagrangian coordinates in the microcirculation.

The cellular composition of the microcirculation creates blood flow that can be unsteady and nonuniform. To obtain information about nonuniform cellular trajectories, we describe in vivo imaging techniques that provide both detailed tracking of individual particles as well as an approach to simultaneous multicolor particle tracking. Particularly relevant to biologic systems, Lagrangian methods provide information about the fate of individual particles and flow in the system.

Intravenous immune globulin prevents venular vaso-occlusion in sickle cell mice by inhibiting leukocyte adhesion and the interactions between sickle erythrocytes and adherent leukocytes.

Sickle cell vaso-occlusion is a complex multistep process likely involving heterotypic interactions among sickle erythrocytes (red blood cells [RBCs]), leukocytes (white blood cells [WBCs]), and endothelial cells. Recent data using intravital microscopy in a sickle cell mouse model suggest that adherent leukocytes in postcapillary venules play a critical role in vaso-occlusion by capturing circulating sickle RBCs. In the course of studies to investigate the adhesion receptors mediating sickle RBC-WBC interactions, we found that control nonspecific immunoglobulin G (IgG) preparations displayed significant inhibitory activity. As a result, we studied the effects of commercial intravenous human immune globulin (i.v.IG) preparations and found that i.v.IG inhibits RBC-WBC interactions in cremasteric venules in a dose-dependent manner. i.v.IG of at least 200 mg/kg dramatically reduced these interactions, even after tumor necrosis factor-alpha (TNF-alpha) stimulation, and not only increased microcirculatory blood flow but also improved survival of sickle cell mice. These data raise the possibility that i.v.IG may have a beneficial effect on sickle cell-associated vaso-occlusion.

Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.

Vascular occlusion is the major cause of morbidity and mortality in sickle cell disease but its mechanisms are poorly understood. We demonstrate by using intravital microscopy in mice expressing human sickle hemoglobin (SS) that SS red blood cells (RBCs) bind to adherent leukocytes in inflamed venules, producing vasoocclusion of cremasteric venules. SS mice deficient in P- and E-selectins, which display defective leukocyte recruitment to the vessel wall, are protected from vasoocclusion. These data uncover a previously unsuspected paradigm for the pathogenesis of sickle cell vasoocclusion in which adherent leukocytes play a direct role and suggest that drugs targeting SS RBC-leukocyte or leukocyte-endothelial interactions may prevent or treat the vascular complications of this debilitating disease.