Cross-circulation and cell distribution kinetics in parabiotic mice.

Blood-borne nucleated cells participate not only in inflammation, but in tissue repair and regeneration. Because progenitor and stem cell populations have a low concentration in the blood, the circulation kinetics and tissue distribution of these cells is largely unknown. An important approach to tracking cell lineage is the use of fluorescent tracers and parabiotic models of cross-circulation. Here, we investigated the cross-circulation and cell distribution kinetics of C57/B6 GFP(+)/wild-type parabionts. Flow cytometry analysis of the peripheral blood after parabiosis demonstrated no evidence for a "parabiotic barrier" based on cell size or surface characterstics; all peripheral blood cell subpopulations in this study reached equilibrium within 14 days. Whole blood fluorescence analysis indicated that the mean exchange flow rate was 16 µl/h or 0.66% of the circulating blood volume per hour. Studies of peripheral lymphoid organs indicated differential cell distribution kinetics. Some subpopulations, such as CD8(+) and CD11c(+), equilibrated in both lymph nodes and spleen indicating a residence time <28 days; in contrast, other lymphocyte subpopulations, such as B220(+) and CD4(+) cells, had not yet reached equilibrium at 28 days. We conclude that parabiosis can provide important insights into defining tissue distribution, residence times, and recirculating pools using fluorochrome markers of cell lineage.

Angiogenesis in wounds treated by microdeformational wound therapy.

BACKGROUND: Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown. OBJECTIVE: To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels. METHODS: Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis. RESULTS: VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiological function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors. CONCLUSIONS: The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

Dynamic determination of oxygenation and lung compliance in murine pneumonectomy.

Thoracic surgical procedures in mice have been applied to a wide range of investigations, but little is known about the murine physiologic response to pulmonary surgery. Using continuous arterial oximetry monitoring and the FlexiVent murine ventilator, the authors investigated the effect of anesthesia and pneumonectomy on mouse oxygen saturation and lung mechanics. Sedation resulted in a dose-dependent decline of oxygen saturation that ranged from 55% to 82%. Oxygen saturation was restored by mechanical ventilation with increased rate and tidal volumes. In the mouse strain studied, optimal ventilatory rates were a rate of 200/minute and a tidal volume of 10 mL/kg. Sustained inflation pressures, referred to as a "recruitment maneuver," improved lung volumes, lung compliance, and arterial oxygenation. In contrast, positive end-expiratory pressure (PEEP) had a detrimental effect on oxygenation; an effect that was ameliorated after pneumonectomy. These results confirm that lung volumes in the mouse are dynamically determined and suggest a threshold level of mechanical ventilation to maintain perioperative oxygen saturation.

Computational flow dynamics in a geometric model of intussusceptive angiogenesis.

Intussusceptive angiogenesis is a process that forms new blood vessels by the intraluminal division of a single blood vessel into two lumens. Referred to as nonsprouting or intussusceptive angiogenesis, this angiogenic process has been described in morphogenesis and chronic inflammation. Mechanical forces are relevant to the structural changes associated with intussusceptive angiogenesis because of the growing evidence that physiologic forces influence gene transcription. To provide a detailed analysis of the spatial distribution of physiologic shear stresses, we developed a 3D finite element model of the intraluminal intussusceptive pillar. Based on geometries observed in adult intussusceptive angiogenesis, physiologic shear stress distribution was studied at pillar sizes ranging from 1 to 10 microm. The wall shear stress calculations demonstrated a marked spatial dependence with discrete regions of high shear stress on the intraluminal pillar and lateral vessel wall. Furthermore, the intussusceptive pillar created a "dead zone" of low wall shear stress between the pillar and vessel bifurcation apex. We conclude that the intraluminal flow fields demonstrate sufficient spatial resolution and dynamic range to participate in the regulation of intussusceptive angiogenesis.

Acellular Dermal Matrix Versus Inferior Deepithelialized Flap Breast Reconstruction: Equivalent Outcomes, with Increased Cost.

BACKGROUND: Approximately 250,000 new cases of breast cancer are diagnosed yearly in the U.S. resulting in more postmastectomy breast reconstructions (PMBRs). The acellular dermal matrix (ADM) expander-implant method became popular in the mid-2000s, but newer techniques such as the inferior deepithelialized flap (IDF) has more recently been described. We hypothesize that ADMs and IDFs provide comparable aesthetic outcomes, with no difference in complication rates and operative characteristics. METHODS: A retrospective, single-institution study was performed between July 1, 2012, and June 30, 2014, examining all PMBR's (ADM and IDF). Outcomes were categorized as clinical (e.g., complications requiring surgical intervention) or aesthetic. RESULTS: A total of 65 patients (41 ADM; 24 IDF; mean age, 53.4 ± 10.7 years) were included, with 101 PMBR's evaluated (63 ADM and 38 IDF). Patients who underwent IDFs had higher body mass index (32 versus 25; P < 0.01) and higher grades of breast ptosis. Major complication rates were similar between ADM and IDF groups (22% versus 31.5%; P = 0.34). There were no differences in aesthetic outcomes between groups (rater intraclass correlation, 0.92). The average IDF breast reconstruction took nearly 30 minutes longer per reconstructed side (192 minutes versus 166 minutes; P = 0.02), but operative costs were more expensive for the ADM breast reconstruction. CONCLUSIONS: The IDF procedure took 30 minutes longer for each reconstructed side, without significant differences in complications or aesthetic outcomes between the 2 PMBRs. IDF reconstructions may be more suitable for patients with grade 3 breast ptosis and higher body mass index. Further studies should focus on long-term outcomes and value-based approaches to PMBR.

A morphometric study of mechanotransductively induced dermal neovascularization.

BACKGROUND: Mechanical stretch has been shown to induce vascular remodeling and increase vessel density, but the pathophysiologic mechanisms and the morphologic changes induced by tensile forces to dermal vessels are poorly understood. METHODS: A custom computer-controlled stretch device was designed and applied to the backs of C57BL/6 mice (n=38). Dermal and vascular remodeling was studied over a 7-day period. Corrosion casting and three-dimensional scanning electron microscopy and CD31 staining were performed to analyze microvessel morphology. Hypoxia was assessed by immunohistochemistry. Western blot analysis of vascular endothelial growth factor (VEGF) and mRNA expression of VEGF receptors was performed. RESULTS: Skin stretching was associated with increased angiogenesis as demonstrated by CD31 staining and vessel corrosion casting where intervascular distance and vessel diameter were decreased (p<0.01). Immediately after stretching, VEGF dimers were increased. Messenger RNA expression of VEGF receptor 1, VEGF receptor 2, neuropilin 1, and neuropilin 2 was increased starting as early as 2 hours after stretching. Highly proliferating epidermal cells induced epidermal hypoxia starting at day 3 (p<0.01). CONCLUSIONS: Identification of significant hypoxic cells occurred after identification of neovessels, suggesting an alternative mechanism. Increased expression of angiogenic receptors and stabilization of VEGF dimers may be involved in a mechanotransductive, prehypoxic induction of neovascularization.

Blood flow shapes intravascular pillar geometry in the chick chorioallantoic membrane.

The relative contribution of blood flow to vessel structure remains a fundamental question in biology. To define the influence of intravascular flow fields, we studied tissue islands--here defined as intravascular pillars--in the chick chorioallantoic membrane. Pillars comprised 0.02 to 0.5% of the vascular system in 2-dimensional projection and were predominantly observed at vessel bifurcations. The bifurcation angle was generally inversely related to the length of the pillar (R = -0.47, P < .001). The pillar orientation closely mirrored the axis of the dominant vessel with an average variance of 5.62 +/- 6.96 degrees (p = .02). In contrast, the variance of pillar orientation relative to nondominant vessels was 36.78 +/- 21.33 degrees (p > .05). 3-dimensional computational flow simulations indicated that the intravascular pillars were located in regions of low shear stress. Both wide-angle and acute-angle models mapped the pillars to regions with shear less than 1 dyn/cm2. Further, flow modeling indicated that the pillars were spatially constrained by regions of higher wall shear stress. Finally, the shear maps indicated that the development of new pillars was limited to regions of low shear stress. We conclude that mechanical forces produced by blood flow have both a limiting and permissive influence on pillar development in the chick chorioallantoic membrane.

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.

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.

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.