Inner Wall Research Articles

Investigating the potential effect of Holothuria scabra extract on osteogenic differentiation in preosteoblast MC3T3-E1 cells

The present medical treatments of osteoporosis come with adverse effects. It leads to the exploration of natural products as safer alternative medical prevention and treatment. The sea cucumber, Holothuria scabra, has commercial significance in Asian countries with rising awareness of its properties as a functional food. This study aims to investigate the effects of the inner wall (IW) extract isolated from H. scabra on extracellular matrix maturation, mineralization, and osteogenic signaling pathways on MC3T3-E1 preosteoblasts. The IW showed the expression of several growth factors. Molecular docking revealed that H. scabra BMP2/4 binds specifically to mammal BMP2 type I receptor (BMPR-IA). After osteogenic induction, the viability of cells treated with IW extract was assessed and designated with treatment of 0.1, 0.5, 1, and 5 µg/ml of IW extract for 21 consecutive days. On days 14 and 21, treatments with IW extract at 1 and 5 µg/ml showed increased alkaline phosphatase (ALP) activity and calcium deposit levels in a dose-dependent manner compared to the control group. Moreover, the transcriptomic analysis of total RNA of cells treated with 5 µg/ml of IW extract exhibited upregulation of TGF-β, PI3K/Akt, MAPK, Wnt and PTH signaling pathways at days 14. This study suggests that IW extract from H. scabra exhibits the potential to enhance osteogenic differentiation and mineralization of MC3T3-E1 preosteoblasts through TGF-β, PI3K/Akt, MAPK, Wnt and PTH signaling pathways. Further investigation into the molecular mechanisms underlying the effect of IW extract on osteogenesis is crucial to support its application as a naturally derived supplement for prevention or treatment of osteoporosis.

Study on the mechanism of rapid densification and structural evolution of C/C composites by cooling pyrolysis

A large thermal gradient about 190 ℃/cm was established in the thickness direction of preform and the cylindrical carbon/carbon composites with radial gradient pyrocarbon (PyC) distribution were rapidly prepared via the cooling pyrolysis technology in the high temperature surface based on the large thermal gradient chemical vapor infiltration (CPTHTS-LTGCVI). The decrease of initial temperature and the increase of cooling rate both contribute to the opening of closed pore and pore expansion, which are conducive to improving the densification degree. Moderate initial temperature (about 1130℃) and cooling rate (10 ℃/5 h) are optimal to rapidly preparing C/C composites with good density uniformity. For the whole cylindrical C/C composites, the faster the cooling rate, the higher the density and density uniformity, the fewer the super macropores. As the cooling rate is 10 ℃/2.5 h, the density of sample can reach 1.57 g/cm3 within 50 h, and the carbon yield in the first stage and accumulation can reach 84 % and 50 %, respectively. From the inner wall (IW) to the outer wall (OW), there are three successive regions about RL, RL+SL and SL PyC with gradually weakened texture, which is related to the insufficient pyrolysis caused by the decreased temperature and increased concentration, and the aromatization caused by the extended gas retention time. The edge dislocations and non-coherent sub-grain boundaries of PyC may be related to the distorted aromatic carbon planes containing five-membered rings. The edge dislocations may be the main reason of texture reduction and structural evolution of PyC.

Atherosclerosis risk assessment in human carotid artery with variation in sinus length: a numerical approach

The mortality rates due to cardiovascular diseases are on a rise globally. One of the major cardiovascular diseases is stroke which occurs due to atherosclerotic plaques build-up in the carotid artery. The common carotid artery (CCA) bifurcates into the internal carotid artery (ICA) and external carotid artery (ECA). Sinus present at ICA is an ellipsoidal-shaped dilated region acting as a pressure receptor and blood flow regulator. Dimensions of the sinus vary from person to person, affecting the hemodynamics of the carotid artery. The current numerical study manifests a 3D flow analysis by varying the sinus length to investigate its local and global effects on the hemodynamics of the carotid artery using various biomechanical risk analysis parameters of atherosclerosis. User-defined function (UDF) dictates the pulsatile flow velocity profile imposed at the inlet. Near the outer wall (OW) of the sinus, the blood flow velocities are lower and recirculation zones are more. Though the recirculation zones for shorter sinus will be close to the inner wall (IW), interestingly, with an increase in the sinus length, the recirculation zones shift toward the OW with higher strength. These significantly decrease the x-wall shear stress (x-WSS) and time-averaged wall shear stress (TAWSS) values on the OW of the longer sinus. The other risk analysis parameters, like oscillatory shear index (OSI) and relative residence time (RRT), support the described consequences. These results reveal that sinus of increased length is more prone to developing atherosclerotic plaque.

Schlemm's Canal Endothelium Cellular Connectivity in Giant Vacuole and Pore Formation in Different Flow-type Areas: A Serial Block-Face Scanning Electron Microscopy Study.

Glaucoma is associated with increased resistance in the conventional aqueous humor (AH) outflow pathway of the eye. The majority of resistance is thought to reside in the juxtacanalicular connective tissue (JCT) region of the trabecular meshwork and is modulated by the inner wall (IW) endothelial cells of Schlemm’s canal (SC). The IW cells form connections with the underlying JCT cells/matrix, and these connections are thought to modulate outflow resistance. Two ways by which AH crosses the IW endothelium are through: 1) the formation of outpouchings in IW cells called giant vacuoles (GVs) and their intracellular pores (I-pores), and 2) intercellular pores between two adjacent IW cells (B-pores). AH outflow is segmental with areas of high-, low-, and non-flow around the circumference of the eye. To investigate whether changes in cellular connectivity play a role in segmental outflow regulation, we used global imaging, serial block-face scanning electron microscopy (SBF-SEM), and 3D reconstruction to examine individual IW cells from different flow areas of ex vivo perfused normal human donor eyes. Specifically, we investigated the differences in cellular dimensions, connections with JCT cells/matrix, GVs, and pores in SC IW cells between high-, low-, and non-flow areas. Our data showed that: 1) IW cell-JCT cell/matrix connectivity was significantly decreased in the cells in high-flow areas compared to those in low- and non-flow areas; 2) GVs in the cells of high-flow areas had significantly fewer connections beneath them compared to GVs in the cells of low- and non-flow areas; 3) Type IV GVs (with I-pores and basal openings) had significantly fewer connections beneath them compared to Type I GVs (no I-pore or basal opening). Our results suggest that a decreased number of cellular connections between the IW and JCT in high-flow areas is associated with increased numbers of GVs with I-pores and larger Type IV GVs observed in previous studies. Therefore, modulating the number of cellular connections may affect the amount of high-flow area around the eye and thereby modulate AH outflow.

Solid-State Nanochannel-Based Sensing Systems: Development, Challenges, and Opportunities

Solid-state nanochannel-based sensing systems with various structures and morphologies have realized precise measurements for various key biomarkers due to their tunable physical structures and morphologies, controllable chemical properties, and a nanoconfined space-induced target enriching effect. In the past several decades, series of solid-state nanochannel-based sensing systems mainly focused on modifying functional elements on nanochannels have allowed for a highly sensitive and specific detection of various key biomarkers between 0.1 and 100 nm, including small molecules, nucleic acids, and proteins. However, traditional solid-state nanochannel-based sensing systems have mainly focused on the functional element modified on their inner-walls (FEIW), ignoring the ion-gating effect of functional elements modified on the outer surface (FEOS). Therefore, the direct detection of targets with sizes larger than the diameters of nanochannels, i.e., cells, was hard to realize. Recently, research has turned its attention to nanochannels with FEOS, which extends the range of measurable biomarkers to cells (50 μm) and promotes precise measurements. In this Perspective, we mainly focus on exhibiting the great breakthroughs of solid-state nanochannels with distinct partitions of the inner wall (IW) and outer surface (OS). Meanwhile, the cutting-edge concept of nanochannels with quantum confined superfluid (QSF) is also discussed. A possible explanation for the ultrafast flow of liquids and gases through nanopores based on wave-particle duality was also provided. The quantum effect on ultrafast flow would provide new perspectives for nanochannel-based sensing systems for various key biomarkers, which may also promote the development of seawater desalination, energy conversion, and information systems.

Morphological factors associated with giant vacuoles with I-pores in Schlemm's canal endothelial cells of human eyes: A serial block-face scanning electron microscopy study

Increased intraocular pressure (IOP) is the main risk factor for primary open-angle glaucoma and results from impaired drainage of aqueous humor (AH) through the trabecular outflow pathway. AH must pass the inner wall (IW) endothelium of Schlemm's canal (SC), which is a monolayer held together by tight junctions, to exit the eye. One route across the IW is through giant vacuoles (GVs) with their basal openings and intracellular pores (I-pores). AH drainage through the trabecular outflow pathway is segmental. Whether more GVs with both basal openings and I-pores are present in the active flow areas and factors that may influence formation of GVs with I-pores have not been fully elucidated due to limitations in imaging methods. In this study, we applied a relatively new technique, serial block-face scanning electron microscopy (SBF-SEM), to investigate morphological factors associated with GVs with I-pores in different flow areas. Two normal human donor eyes were perfused at 15 mmHg with fluorescent tracers to label the outflow pattern followed by perfusion-fixation. Six radial wedges of trabecular meshwork including SC (2 each from high-, low-, and non-flow areas) were imaged using SBF-SEM (total: 9802 images). Total GVs, I-pores, basal openings, and four types of GVs were identified. Percentages of GVs with I-pores and basal openings and number of I-pores/GV were determined. Overall, 14.4% (477/3302) of GVs had I-pores. Overall percentage of GVs with both I-pores and basal openings was higher in high- (15.7%), than low- (12.6%) or non-flow (7.3%) areas. Of GVs with I-pores, 83.2% had a single I-pore; 16.8% had multiple I-pores (range: 2–6). Additionally, 180 GVs (90 with I-pores and 90 without I-pores) were randomly selected, manually segmented, and three-dimensionally (3D) reconstructed to determine size, shape, and thickness of the cellular lining. Size of GVs (including median volume, surface area, and maximal cross-sectional area) with I-pores (n = 90) was significantly larger than GVs without I-pores (n = 90) using 3D-reconstructed GVs (P ≤ 0.01). Most I-pores (73.3%; 66/90) were located on or close to GV's maximal cross-sectional area with significant thinning of the cellular lining. Our results suggest that larger size and thinner cellular lining of GVs may contribute to formation of GVs with I-pores. More GVs with I-pores and basal openings were observed in high-flow areas, suggesting these GVs do provide a channel through which AH passes into SC and that increasing this type of GV may be a potential strategy to increase aqueous outflow for glaucoma treatment.

Study of microstructure and mechanical property heterogeneity throughout the wall thickness of high strength aluminum alloy thick-wall pipe

Abstract

The Role of Schlemm's Canal Endothelium Cellular Connectivity in Giant Vacuole Formation: A 3D Electron Microscopy Study.

PurposeWe investigated whether cellular connectivity between Schlemm's canal (SC) inner wall (IW) endothelium, and juxtacanalicular connective tissue (JCT), and between IW endothelial cells, plays a role in giant vacuole (GV) and pore formation by comparing perfusion- and immersion-fixed eyes.MethodsNormal human donor eyes (n = 4) were either immersion-fixed (0 mm Hg) or perfusion-fixed (15 mm Hg). Trabecular meshwork near SC was imaged using serial block-face scanning electron microscopy. A total of 12 IW cells from each group were 3D-reconstructed from ∼7040 electron micrographs and compared. In each cell, connections between IW cells and JCT cells/matrix were quantified; IW/IW connectivity was measured by cell border overlap length. GV volume, density, shape, and intracellular and paracellular pores were analyzed.ResultsThe mean number of IW/JCT cell-cell connections per cell significantly decreased (P < 0.01) while the summed GV volume per cell significantly increased (P < 0.01) in perfusion-fixed eyes compared to immersion-fixed eyes. Intracellular pores were observed in 14.6% of GVs in perfusion-fixed eyes and not observed in immersion-fixed eyes. The mean IW/IW overlap length per cell decreased (P < 0.01), and paracellular pores were found only in regions where IW/IW connectivity was minimal (overlap length = 0 μm) in perfusion-fixed eyes and not observed in immersion-fixed eyes.ConclusionsOur data suggest that changes in IW/JCT connectivity may be an important factor in the formation of larger GVs, and decreased IW/IW connectivity may promote paracellular pore formation. Targeting the IW/JCT and IW/IW connectivity may therefore be a potential strategy to regulate outflow resistance and IOP.

Distinct functional elements for outer-surface anti-interference and inner-wall ion gating of nanochannels

Over the decades, widespread advances have been achieved on nanochannels, including nanochannel-based DNA sequencing, single-molecule detection, smart sensors, and energy transfer and storage. However, most interest has been focused on the contribution from the functional elements (FEs) at the inner wall (IW) of nanochannels, whereas little attention has been paid to the contribution from the FEs at the nanochannels’ outer surface (OS). Herein, we achieve explicit partition of FEOS and FEIW based on accurate regional-modification of OS and IW. The FEIW are served for ionic gating, and the chosen FEOS (hydrophobic or charged) are served for blocking interference molecules into the nanochannels, decreasing the false signals for the ionic gating in complex environments. Furthermore, we define a composite factor, areas of a radar map, to evaluate the FEOS performance for blocking interference molecules.

Imaging collector channel entrance with a new intraocular micro-probe swept-source optical coherence tomography.

To describe the use of a newly developed side-viewing catheter probe to provide the cross-sectional images of collector channel entrance (CCE), achieved by swept-source optical coherence tomography (SS-OCT). A side-viewing SS-OCT catheter probe was developed that has a core probe diameter of 0.15mm and an outer diameter of 0.25mm, for the purpose of imaging CCEs within eye globe. Cadaver eyes harvested from swine and human were used to demonstrate its feasibility. For porcine eyes, the probe imaged the CCE by accessing the region of the aqueous plexus (AP) as well as along the inner wall (IW) of the trabecular meshwork (TM). For human eyes, the CCE images were captured by placing the probe within the lumen of the Schlemm's canal (SC) and along its IW. With the optical coherence tomography (OCT) catheter probe, the CCE is well delineated as optically empty areas within the highly scattering sclera. In porcine eyes, images captured in the region of the AP demonstrate a large cavity with delicate tissue strands around the probe. The CCE can be identified at the outer margin of the AP. When imaged along the IW, the TM is discernable but difficult to be distinguished from the AP. In the human limbal regions, when placed within the lumen of the SC, the catheter probe fully occupies the potential space. TM is highly compact. The CCE can be identified at the outer wall of the SC. When imaged along the IW of TM, the SC and CCE can be identified. The intraocular SS-OCT catheter probe is feasible to provide the CCE images, indicating useful clinical applications to assist glaucoma surgery.

Netarsudil Increases Outflow Facility in Human Eyes Through Multiple Mechanisms.

PurposeNetarsudil is a Rho kinase/norepinephrine transporter inhibitor currently in phase 3 clinical development for glaucoma treatment. We investigated the effects of its active metabolite, netarsudil-M1, on outflow facility (C), outflow hydrodynamics, and morphology of the conventional outflow pathway in enucleated human eyes.MethodsPaired human eyes (n = 5) were perfused with either 0.3 μM netarsudil-M1 or vehicle solution at constant pressure (15 mm Hg). After 3 hours, fluorescent microspheres were added to perfusion media to trace the outflow patterns before perfusion-fixation. The percentage effective filtration length (PEFL) was calculated from the measured lengths of tracer distribution in the trabecular meshwork (TM), episcleral veins (ESVs), and along the inner wall (IW) of Schlemm's canal after global and confocal imaging. Morphologic changes along the trabecular outflow pathway were investigated by confocal, light, and electron microscopy.ResultsPerfusion with netarsudil-M1 significantly increased C when compared to baseline (51%, P < 0.01) and to paired controls (102%, P < 0.01), as well as significantly increased PEFL in both IW (P < 0.05) and ESVs (P < 0.01). In treated eyes, PEFL was significantly higher in ESVs than in the IW (P < 0.01) and was associated with increased cross-sectional area of ESVs (P < 0.01). Percentage effective filtration length in ESVs positively correlated with the percentage change in C (R2 = 0.58, P = 0.01). A significant increase in juxtacanalicular connective tissue (JCT) thickness (P < 0.05) was found in treated eyes compared to controls.ConclusionsNetarsudil acutely increased C by expansion of the JCT and dilating the ESVs, which led to redistribution of aqueous outflow through a larger area of the IW and ESVs.

Variations in active outflow along the trabecular outflow pathway

Previous tracer studies have shown segmental outflow in the trabecular meshwork (TM) and along the inner wall (IW) of Schlemm's canal (SC). Whether segmental outflow is conserved distal to SC has not yet been investigated. This study aims to investigate whether the segmented pattern of outflow is conserved in distal outflow pathways by using a newly developed global imaging method and to evaluate variations of active outflow in three distinct regions along trabecular outflow pathway.Six normal whole globe human eyes were first perfused at 15 mmHg to establish a stable baseline outflow facility. The anterior chamber was then exchanged (5 mL) and perfused with fluorescent microspheres (0.002% v/v, 200 μL) to label areas of active outflow. All eyes were perfusion fixed and dissected into anterior segments. The TM and scleral surface were en face imaged globally. Effective filtration area (EFA) and fluorescent tracer distribution and intensity were analyzed in global images for both the TM and episcleral veins (EPVs). Anterior segments were further dissected into a minimum of 16 radial wedges, from which frontal sections were cut, stained, and imaged, using confocal microscopy. EFA from all three locations along the trabecular outflow pathway were measured and compared. Additionally, TM thickness, SC height, and total number of collector channels (CC) were analyzed and compared between active and inactive areas of outflow. Statistical analysis was performed using Student's t-tests and Wilcoxon signed-rank test with a required significance of p ≤ 0.05.All three locations showed a segmental outflow pattern. The TM had a significantly higher mean EFA (86.3 ± 3.5%) compared to both the IW (34.7 ± 2.9%; p ≤ 0.01) and EPVs (41.1 ± 3.8%; p ≤ 0.01). No significant difference in mean EFA was found between IW and EPVs. Preferential active outflow was observed in the nasal and inferior quadrants. TM thickness was significantly larger in areas of active outflow (103.3 ± 4.0 μm; p ≤ 0.01) compared to areas of inactive outflow (78.5 ± 6.5 μm), but there was no significant difference in SC height between active and inactive outflow areas. Among all eyes, a total of 80 CCs were counted with 63 associated with active outflow and 17 associated with inactive outflow. A higher number of CCs associated with areas of active outflow were found in the nasal (26 of 63) and inferior (20 of 63) quadrants compared to the temporal (9 of 63) and superior (8 of 63) quadrants.A segmental nature of outflow is conserved along the trabecular outflow pathway with variations in three distinct locations (TM, IW, and EPVs). IW and EPVs showed a similar mean EFA. Preferential active outflow was observed in the nasal and inferior quadrants of the eye, which are associated with more expanded TM and higher number of CCs. Normal outflow patterns and its variations along the outflow pathway reported in this study will provide the basis for future studies of the outflow changes in eyes with glaucoma.

Characterization of scrape-off layer transport in JET limiter plasmas

The JET scrape-off layer (SOL) has been characterized with a reciprocating probe in inner wall (IW), and outer wall (OW), limited plasmas. Experiments revealed that SOL profiles are substantially broader (by a factor of ∼5–7.5 in the power e-folding length) for IW limited than in OW limited plasmas. Results are consistent with the larger radial turbulent transport found for IW limited plasmas. Major differences are observed between IW and OW limited plasmas on the density and electron temperature e-folding lengths, parallel flow, radial turbulent transport as well as on the temporal and spatial characteristics of the fluctuations. Experimental findings on JET suggest that the differences in the SOL characteristics for both configurations are due to a combination of a poloidal asymmetry in radial transport with a reduced cross-field transport across the last closed flux surface associated with the confinement improvement observed for OW limited plasmas.The dependence of the SOL power e-folding length on the main plasma parameters was also investigated for IW limited plasmas and a modest negative dependence on both the plasma current and the line-averaged density found. Finally, it is shown that the SOL radial transport and the amplitude of the fluctuations increase with plasma current and decrease with line-averaged density for IW limited plasmas.

Reversible changes in aqueous outflow facility, hydrodynamics, and morphology following acute intraocular pressure variation in bovine eyes

Background Elevated intraocular pressure (IOP) is primarily due to increased aqueous outflow resistance, but how aqueous outflow resistance is generated and regulated are still not fully understood. The aim of this study is to determine whether changes in outflow facility, outflow pattern, and morphology following acute IOP elevation were reversible when the IOP was returned to a normal level in bovine eyes using a two-color tracer technique to label outflow patterns within the same eye. Methods Twelve fresh enucleated bovine eyes were perfused with Dulbecco's phosphate buffer saline (PBS) containing 5.5 mmol/L glucose (DBG) at 30 mmHg first to establish the baseline outflow facility followed by a fixed volume of red fluorescent microspheres (0.5 μm, 0.002% v/v). After the red tracer being replaced with DBG in the anterior chamber, perfusion was continued at 7 mmHg with the same volume of green tracer, followed by a fixative. In two control groups, the eyes were constantly perfused at either 30 mmHg (n=6) or 7 mmHg (n=6) using the same methods. The outflow facility (C, μl·min·-1mmHg-1), was continuously recorded. Confocal images were taken along the inner wall (IW) of the aqueous plexus (AP) in frontal sections. The percent of the effective filtration length (PEFL, PEFL=IW length exhibiting tracer labeling/total length of IW) was measured. Sections with AP were processed and examined by light microscopy. The total length of IW and the length exhibiting separation (SL) in the juxtacanalicular connective tissue (JCT) were measured. A minimum of eight collector channel (CC) ostia per eye were analyzed for herniations. Results In the experimental (30-7 mmHg) group, the outflow facility was significantly higher at 7 mmHg ((4.81±1.33) μl·min·-1mmHg-1) than that at 30 mmHg ((0.99±0.15) μl·min·-1mmHg-1, P=0.002), corresponding to a significant increase in the PEFL (P=0.0003). The percent of CC ostia exhibiting herniations in the experimental group ((67.40±8.90) μl·min·-1mmHg-1) decreased significantly compared to that in the control at 30 mmHg ((94.44±3.33) μl·min·-1mmHg-1, P=0.03), but higher than that in the control at 7 mmHg ((29.43±4.60) μl·min·-1mmHg-1, P=0.01). Washout-associated separation between the IW and JCT was found by light microscopy and percent separation length (PSL, PSL=SL/total length of IW) was decreased in the control at 30 mmHg compared to that in the experimental group and control at 7 mmHg. Conclusions The pressure-induced morphological and hydrodynamic changes were reversible. Changes (collapse of AP, separation between the JCT and IW, and herniation into CC ostia) influence the effective filtration area that regulates outflow facility.

Similar hydrodynamic and morphological changes in the aqueous humor outflow pathway after washout and Y27632 treatment in monkey eyes

Our previous studies in bovine eyes demonstrated that the structural correlate to the increase in outflow facility after either Rho-kinase inhibitor Y-27632 (Y27) treatment or washout appeared to be separation between the juxtacanalicular tissue (JCT) and inner wall (IW) of the aqueous plexus, the bovine equivalent of Schlemm’s canal (SC). While these findings suggest that Y27 and washout may increase outflow facility through a similar mechanism, the anatomy of bovine outflow pathway differs considerably from both the human and monkey outflow pathway; however, only the human eye does not exhibit washout. In light of this, we compared the effects of Y27 and washout on outflow facility, hydrodynamic patterns of outflow, and the morphology of the IW and JCT in monkey eyes, given that their anatomy is closer to human eyes. Twelve freshly enucleated monkey eyes were used in this study. Eyes were perfused with Dulbecco’s PBS containing 5.5 mM glucose (GPBS) to establish a baseline facility at 15 mmHg. Four eyes were perfused for a short-duration (30 min) as a control, 4 eyes for a long-duration (180 min) to induce washout, and 4 eyes with GPBS+50 μM Y27 for 30 min. All eyes were then perfused with fluorescent microspheres (0.5 μm; 0.002%) to label the hydrodynamic patterns of outflow and then perfusion-fixed. Confocal images of frontal sections were taken along the IW of SC. The total length (TL) and the tracer-decorated length (FL) of the IW were measured to calculate the average percent effective filtration length (PEFL = FL/TL). Sections with SC were examined by light and electron microscopy. The TL of the IW and the length exhibiting separation (SL) in the JCT were measured to calculate the average percent separation length (PSL = SL/TL). Outflow facility increased 149.2% ( p < 0.01) from baseline after washout during long-duration perfusion, and 114.9% ( p = 0.004) after Y27 treatment, but did not change significantly after short-duration perfusion in control eyes ( p = 0.46). Distribution of the tracer labeling appeared punctate along the IW of control eyes, while a more uniform pattern was observed after washout and Y27 treatment. PEFL in washout (83.4 ± 2.1%) and Y27 treated eyes (82.5 ± 1.6%) was 3.4-fold larger compared to controls (24.2 ± 4.2%, P < 0.001). The JCT appeared distended with loss of connections between JCT cells and between JCT cells and their extracelluar matrix in eyes with washout or after Y-27 treatment. PSL in the JCT was 2.3-fold larger in washout eyes (77.4 ± 3.3%) and 2.2-fold larger in Y27 treated eyes (75.2 ± 5.3%) versus controls (33.5 ± 5.3%, p = 0.001). Significant positive correlations were found between outflow facility and PEFL, facility and PSL and between PEFL and PSL. Our data demonstrated that similar hydrodynamic and morphological changes occurred in the aqueous humor outflow pathway of monkey eyes after induction of washout and Y27 treatment. Both Y27 and washout increase outflow facility by redistributing aqueous outflow through a larger area in the JCT. These hydrodynamic changes are likely driven by morphologic changes associated with a decrease in cell–cell and cell–matrix connections in the JCT.

Reduced intercarotid artery distance in acromegaly: pathophysiologic considerations and implications for transsphenoidal surgery

Background The objective of this study is to evaluate the significance of reduced intercarotid artery distance in the C5 segment in acromegalic patients and the implications for transsphenoidal surgery. Methods The skull base with the carotid canal was examined with a helical CT scan in 45 patients with acromegaly and 45 age group–matched controls. The distances between the inner walls (IWs) of the carotid sulcus (CS), the outer walls (OWs) of the CS, and the diameter of the ICA at the C5 segment were assessed. Preoperative IGF-1 and growth hormone levels and concomitant diseases were evaluated and correlated with the biometric findings. Statistical analysis was performed with JMP (JMP version 7.0.2, SAS, Cary, USA). Results The mean distance between the IW was 1.64 ± 0.40 cm in the acromegalic patients and 1.90 ± 0.26 cm in the control group ( P = .0005). The distance between the OW measured 3.01 ± 0.39 and 2.97 ± 0.33 cm in the acromegalics and in the control group, respectively ( P = .6230). The difference in the diameter of the ICA was statistically significant ( P < .0001) between patients and control group. Within the patient group, the distance between the IW of both ICA was significantly smaller in the subgroup with arterial hypertension ( P = .0256). Conclusion Narrowing of the inner borders of the CS between the right and left side is a statistically significant parameter in acromegaly. Attention should be given to an altered vascular course of the ICAs when planning and performing transsphenoidal microsurgery in acromegalic patients. A preoperative skull base CT may furnish important anatomical information and further reduce the risk of vascular injury.

Relationships between increased aqueous outflow facility during washout with the changes in hydrodynamic pattern and morphology in bovine aqueous outflow pathways

Previous studies suggest that the structural correlate for the increased outflow facility (C) during washout in the bovine eye is separation between the inner wall (IW) and underlying juxtacanalicular connective tissue (JCT). However, how these structural changes affect hydrodynamic patterns of outflow during washout has not been studied. We hypothesize that an increase in the outflow facility during washout is associated with an increase in the effective filtration area (EFA) of aqueous outflow, which is regulated by a loss of the connectivity between the IW and JCT. To test this hypothesis, the relationship between C and the hydrodynamic patterns of outflow as well as the morphological changes in JCT and IW during the washout were investigated. Ten bovine eyes were perfused at 15 mmHg with Dulbecco's PBS + 5.5 mM glucose (DPBS) for 30 min to establish stable baseline C. After measuring baseline C, five eyes ( short-duration group) were perfused with 0.5 mL DPBS containing 0.002% microspheres (0.5 μm) to trace the hydrodynamic pattern of outflow. Five other eyes ( long-duration group) were perfused for 3 h to elicit a significant washout effect followed by subsequent perfusion of the same volume (0.5 mL) of microspheres to map out the outflow pattern after washout. All eyes were then perfusion-fixed. Anterior segments were sectioned and prepared for confocal and light microscopy. Total length (TL) and filtration length (FL) of the IW were measured in ≥15 images/eye to calculate percent effective filtration length (PEFL = FL/TL) while TL and length exhibiting JCT/IW separation (SL) were measured in ≥13 images/eye to calculate percent separation length (PSL = SL/TL). In long-duration eyes, C increased 170.5 ± 21.3% (mean ± SEM, 1.55 ± 0.24 vs 4.13 ± 0.55 μl/min/mmHg, p = 0.001) above baseline. Pre-fixation C (4.13 ± 0.55 μl/min/mmHg) in long-duration was 1.6-fold greater than that (2.14 ± 0.61 μl/min/mmHg; p = 0.042) in short-duration. A more uniform tracer labeling was observed in the JCT/IW of long-duration eyes compared to short-duration. PEFL was 2.3-fold larger (52.82 ± 6.06 vs. 22.2 ± 6.0%; p = 0.007) and PSL was 2.6-fold larger (54.2 ± 6.0 vs. 20.5 ± 1.3%; p = 0.004) in long-duration eyes compared to short-duration. Data from all eyes revealed a positive correlation between PEFL and PSL ( p = 0.02). Both PEFL and PSL demonstrated significant positive correlations with the relative increase in C due to washout ( p ≤ 0.05). An additional experiment was performed in which unequal volumes of tracer (0.5 and 1.0 mL) were perfused in paired eyes for both short- and long-duration ( N = 2 for each condition) to examine the affect on PEFL. No significant change in PEFL was found in eyes perfused with 0.5 and 1.0 mL within the same group. These data support our hypothesis that separations between the IW and JCT result in an increase in the EFA that then influences C. Altogether, these data suggest that outflow hydrodynamics and the tissue structure work together to regulate outflow resistance.

Morphological and hydrodynamic correlates in monkey eyes with laser induced glaucoma

This study investigated the relationship between decreased outflow facility (C) and changes in hydrodynamic aqueous humor outflow patterns and morphology in cynomolgus monkey eyes with unilateral chronically elevated intraocular pressure (IOP). Argon laser photocoagulation burns to the trabecular meshwork (TM) were made in one eye of each monkey (N = 3), leaving the contralateral eye as a normotensive control. IOPs were followed by pneumatonometry for 16–70 months. C was measured by fluorophotometry before sacrifice. To label the hydrodynamic patterns of outflow, the eyes were enucleated and perfused with fluorescent microspheres (0.5 μm; 0.002%) at the last pressure measured before death minus 7 mmHg. The eyes were perfusion-fixed at the same pressure. Confocal images were taken along the inner wall (IW) of the Schlemm's canal (SC). The total length (TL) and the filtration length (FL) of the IW decorated by tracers were measured in frontal sections. The average percent effective filtration length (PEFL = FL/TL) was calculated for each eye. Sections exhibiting SC were processed and examined under light and electron microscopy. The average IOP was significantly higher in laser-treated eyes (mean ± SD = 61.33 ± 4.16 mmHg) than controls (22.67 ± 4.16 mmHg, P = 0.002). The average C was 13-fold lower in laser-treated eyes (0.03 ± 0.02 μl/min/mmHg) than controls (0.39 ± 0.17 μl/min/mmHg, P = 0.057). By confocal microscopy, in control eyes, SC was open and a segmental distribution of microspheres was found in the TM with a greater concentration near the collector channel (CC) ostia. Much less tracer labeling was seen along SC in laser-treated eyes than control eyes. The average PEFL in controls (47.47 ± 10.79%) was 6-fold larger than in laser-treated eyes (8.40 ± 4.81%, P = 0.048). The average distance between the inner and outer wall of SC was 5-fold greater in control eyes (18.99 ± 6.03 μm) than in laser-treated eyes (3.47 ± 0.33 μm, P = 0.048). By light microscopy, there was extensive pigmentation throughout the TM, denser extracellular matrix in the JCT region, and most of SC collapsed with focal herniations of the IW and JCT protruding into the CC ostia in laser-treated eyes. By electron microscopy, few or no microspheres were observed in laser-treated areas and the areas with SC collapse. More microspheres were observed near the CC ostia area in non-lasered areas. In conclusion, in the laser-induced glaucoma model, laser damage results in a reduction in the available area for outflow across the IW of SC which contributes to the decrease in C and thus elevation of the IOP. Constriction of SC, caused by the chronic elevation of IOP, further decreases the available area for outflow across the IW which decreases C even more in a vicious cycle. This study suggests that the available area for aqueous humor outflow across the IW of SC may play a role in regulating outflow resistance and maintaining IOP.

Reduction of the Available Area for Aqueous Humor Outflow and Increase in Meshwork Herniations into Collector Channels Following Acute IOP Elevation in Bovine Eyes

To understand how hydrodynamic and morphologic changes in the aqueous humor outflow pathway contribute to decreased aqueous humor outflow facility after acute elevation of intraocular pressure (IOP) in bovine eyes. Enucleated bovine eyes were perfused at 1 of 4 different pressures (7, 15, 30, 45 mm Hg) while outflow facility was continuously recorded. Dulbecco PBS + 5.5 mM glucose containing fluorescent microspheres (0.5 mum, 0.002% vol/vol) was perfused to outline aqueous outflow patterns, followed by perfusion-fixation. Confocal images were taken along the inner wall (IW) of the aqueous plexus (AP) in radial and frontal sections. Percentage effective filtration length (PEFL; IW length exhibiting tracer labeling/total length of IW) was measured. Herniations of IW into collector channel (CC) ostia were examined and graded for each eye by light microscopy. Increasing IOP from 7 to 45 mm Hg coincided with a twofold decrease in outflow facility (P < 0.0001), a 33% to 57% decrease in PEFL with tracer confined more to the vicinity of CC ostia, progressive collapse of the AP, and increasing percentage of CC ostia exhibiting herniations (from 15.6% +/- 6.5% at 7 mm Hg to 95% +/- 2.3% at 30 mm Hg [P < 10(-4)], reaching 100% at 45 mm Hg). Decreasing outflow facility during acute IOP elevation coincides with a reduction in available area for aqueous humor outflow and the confinement of outflow to the vicinity of CC ostia. These hydrodynamic changes are likely driven by morphologic changes associated with AP collapse and herniation of IW of AP into CC ostia.

The mechanism of increasing outflow facility by rho-kinase inhibition with Y-27632 in bovine eyes

Rho-kinase inhibitor Y-27632 (Y-27) affects actomyosin cytoskeletal networks and has been shown to significantly increase outflow facility (C) in enucleated porcine and rabbit eyes, as well as in vivo monkey eyes without obvious toxicity. The mechanisms underlying these responses remain largely unknown. In this study, we investigate how Y-27 affects aqueous humor C, the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular connective tissue (JCT). 12 bovine eyes were perfused at 15mmHg with Dulbecco's PBS containing 5.5mM glucose (DPBS) to establish stable baseline C. The anterior chamber was exchanged and perfused with DPBS containing 50μM Y-27 in 7 eyes, while 5 eyes received DPBS alone. Eyes were then perfused with DPBS containing fluorescent microspheres (0.5μm; 0.002% v/v) at a fixed volume to deliver equivalent amounts of tracer to label the hydrodynamic filtration patterns. All eyes were perfusion-fixed with Karnovsky's fixative. Radial and frontal sections were prepared in all quadrants and confocal images were taken along the IW of the aqueous plexus (AP). The total length (TL) and filtration length (FL) of the IW were measured in ≥16 images/eye, and the average percent effective filtration length (PEFL=FL/TL) was calculated. Sections with AP were processed and examined by light and electron microscopy. The TL of the IW and length exhibiting JCT/IW separation (SL) were measured in ≥16 micrographs/eye, and the average percent separation length (PSL=SL/TL) was also calculated. After Y-27 treatment, C increased from 1.54±0.34 (±SEM) to 2.36±0.54μL/min per mmHg (58.2±18.9%) while control eyes changed from 1.67±0.41 to 1.71±0.39μl/min per mmHg (6.0±9.3%) and the percent changes between the Y-27-treated and control eyes were significant (p=0.03). Control eyes showed segmental distribution of tracer in the trabecular meshwork tending to cluster near collector channel ostia, whereas Y-27-treated eyes showed a more uniform pattern and extensive labeling along the IW. In Y-27-treated eyes, PEFL was 3-fold larger (57.6±3.7%) compared to control eyes (18.2±4.5%; p<0.001). Light microscopic examination revealed that, with Y-27, the IW and JCT were significantly distended compared to control eyes, with discernible separation between the IW and JCT. PSL was 2.8-fold larger in Y-27-treated eyes (59.3±3.6%) than in controls (20.8±2.0%; p<0.001). A significant positive correlation was found between PEFL and PSL (p=0.003) suggesting that as connectivity between the JCT and IW decreases the available area for aqueous humor drainage increases along the AP. Our study also demonstrates a significant positive correlation between C and the PSL (p=0.01), a finding identical to what we reported to occur during the “washout” effect in bovine eyes. Our data suggests the structural correlate to the increase in C and PEFL after Y-27-treatment is physical separation between the JCT and IW. The increase in C after Y-27-treatment may share a similar mechanism comparable with the washout effect that occurs in bovine eyes. Overall, these findings support our hypothesis that JCT/IW connectivity influences local outflow hydrodynamics that regulate C, and suggest that strategies targeting JCT/IW connectivity are promising anti-glaucoma therapies to reduce IOP.