Aslihan Turhan

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. Anat Rec, 2010.

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-β-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.

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 μm vs. 28 ± 16 μm), 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. Anat Rec, 291:74–82, 2007.

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.

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.

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. Microsc. Res. Tech., 2009.

Trafficking of Immune Cells in the Cornea and Ocular Surface

The role of immuno-inflammatory responses in the cornea and ocular surface has continuously been evolving over the past decades and has been becoming the center stage for therapeutic approaches for many diseases. In fact, the relevance of inflammation as a significant component in the pathophysiology of most acute and chronic forms of corneal and ocular surface diseases (e.g., microbial keratitis, allergy, and dry eye syndrome) has become evident. Both local and systemic immunomodulation with anti-inflammatory agents have been used successfully in improving these conditions or bringing these conditions under control. Thus, understanding the cellular and molecular mechanisms by which the ocular surface participates in immuno-inflammatory disorders is crucial for a more rational clinical approach to treating these diseases.

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.

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 μm diameter) involving six to ten capillaries with a luminal diameter of 6.8 ± 1.1 μm. 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 μm/sec within the feeding mucosal plexus to 491 ± 155 μm/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. Anat Rec, 292:621–632, 2009.