Discussion: "Comparison of Statistical Methods for Assessing Spatial Correlations Between Maps of Different Arterial Properties" (Rowland, E. M., Mohamied, Y., Chooi, K. Y., Bailey, E. L., and Weinberg, P. D., 2015, ASME J. Biomech. Eng., 137(10), p. 101003): An Alternative Approach Using Segmentation Based on Local Hemodynamics.

The biological response of living arteries to mechanical forces is an important component of the atherosclerotic process and is responsible, at least in part, for the well-recognized spatial variation in atherosusceptibility in man. Experiments to elucidate this response often generate maps of force and response variables over the arterial surface, from which the force-response relationship is sought. Rowland et al. discussed several statistical approaches to the spatial autocorrelation that confounds the analysis of such maps and applied them to maps of hemodynamic stress and vascular response obtained by averaging these variables in multiple animals. Here, we point out an alternative approach, in which discrete surface regions are defined by the hemodynamic stress levels they experience, and the stress and response in each animal are treated separately. This approach, applied properly, is insensitive to autocorrelation and less sensitive to the effect of confounding hemodynamic variables. The analysis suggests an inverse relation between permeability and shear that differs from that in Rowland et al. Possible sources of this difference are suggested.

All-trans retinoic Acid regulates cx43 expression, gap junction communication and differentiation in primary lens epithelial cells.

PURPOSE: To examine the effect of all-trans retinoic acid (ATRA) treatment on connexin 43 (Cx43) expression, gap junction intercellular communication (GJIC), and cellular differentiation in primary canine lens epithelial cells (LEC). METHODS AND MATERIALS: Dose and time-dependent effects of ATRA on Cx43 protein, mRNA and GJIC, were assessed by immunoblotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and scrape loading/dye transfer assays, respectively. Expression of beta crystallin was evaluated by immunoblotting. RESULTS: Treatment with ATRA at non-cytotoxic concentrations significantly increased Cx43 protein, mRNA and GJIC in primary canine LEC. Treatment with ATRA for five and seven days increased levels of beta crystallin, a protein marker of LEC differentiation. Inhibition of GJIC via pre-treatment with a synthetic inhibitor, 18-alpha glycyrrethinic acid (AGA), reduced ATRA-induced increases in Cx43 and GJIC and partially blocked ATRA-induced beta crystallin protein. CONCLUSIONS: Treatment with ATRA significantly increased Cx43 expression and GJIC in canine LEC, and these effects were associated with increased LEC differentiation. Results from this study suggest that functional gap junctions may play a role in the modulation of cellular differentiation in primary canine LEC.

Cerebrospinal fluid dynamics in the human cranial subarachnoid space: an overlooked mediator of cerebral disease. I. Computational model.

Abnormal cerebrospinal fluid (CSF) flow is suspected to be a contributor to the pathogenesis of neurodegenerative diseases such as Alzheimer's through the accumulation of toxic metabolites, and to the malfunction of intracranial pressure regulation, possibly through disruption of neuroendocrine communication. For the understanding of transport processes involved in either, knowledge of in vivo CSF dynamics is important. We present a three-dimensional, transient, subject-specific computational analysis of CSF flow in the human cranial subarachnoid space (SAS) based on in vivo magnetic resonance imaging. We observed large variations in the spatial distribution of flow velocities with a temporal peak of 5 cm s(-1) in the anterior SAS and less than 4 mm s(-1) in the superior part. This could reflect dissimilar flushing requirements of brain areas that may show differences in susceptibility to pathological CSF flow. Our methods can be used to compare the transport of metabolites and neuroendocrine substances in healthy and diseased brains.

Cerebrospinal fluid dynamics in the human cranial subarachnoid space: an overlooked mediator of cerebral disease. II. In vitro arachnoid outflow model.

The arachnoid membrane (AM) and granulations (AGs) are important in cerebrospinal fluid (CSF) homeostasis, regulating intracranial pressure in health and disease. We offer a functional perspective of the human AM's transport mechanism to clarify the role of AM in the movement of CSF and metabolites. Using cultures of human AG cells and a specialized perfusion system, we have shown that this in vitro model mimics the in vivo characteristics of unidirectional fluid transport and we present the first report of serum-free permeability values (92.5 microl min(-1) mm Hg(-1) cm(-2)), which in turn are in agreement with the CSF outflow rates derived from a dynamic, in vivo magnetic resonance imaging-based computational model of the subarachnoid cranial space (130.9 microl min(-1) mm Hg(-1) cm(-2)). Lucifer yellow permeability experiments have verified the maintenance of tight junctions by the arachnoidal cells with a peak occurring around 21 days post-seeding, which is when all perfusion experiments were conducted. Addition of ruthenium red to the perfusate, and subsequent analysis of its distribution post-perfusion, has verified the passage of perfusate via both paracellular and transcellular mechanisms with intracellular vacuoles of approximately 1 microm in diameter being the predominant transport mechanism. The comparison of the computational and in vitro models is the first report to measure human CSF dynamics functionally and structurally, enabling the development of innovative approaches to modify CSF outflow and will change concepts and management of neurodegenerative diseases resulting from CSF stagnation.

Cerebrospinal fluid outflow: an evolving perspective.

Cerebrospinal fluid (CSF) serves numerous important functions in the central nervous system. Despite numerous reports characterizing CSF and its circulation in the subarachnoid space, our understanding of CSF outflow remains limited. Although initial work suggested that both arachnoid granulations and lymphatic capillaries shared in the role of CSF outflow, predominant work since then has focused on the arachnoid granulations. A growing body of recent evidence not only suggests the importance of both arachnoid granulations and lymphatic capillaries, but also additional contributions through transependymal passage likely share in the role of CSF outflow. Consideration of all mechanisms and pathways will help us to better understand the significance of CSF outflow, in health and disease. Here we review how the present concept of CSF outflow has evolved, including a historical review of significant findings and a discussion of the latest innovative developments.

Ex vivo model of cerebrospinal fluid outflow across human arachnoid granulations.

PURPOSE: The brain's arachnoid membrane with granulations is an important biological barrier whose responsibilities include the transmission of cerebrospinal fluid (CSF) and the regulation of pressure. Membrane disturbance may cause changes that are difficult to replicate with animal models, suggesting the need for a model using human arachnoid membrane with granulations for the study of conditions such as Alzheimer disease, hydrocephalus, and pseudotumor cerebri. The authors detail the development and validation of an ex vivo model of CSF outflow across human arachnoid granulations (AGs) as an approximation of in vivo conditions. METHODS: Human AGs were perfused at normal physiological pressure in physiological and nonphysiological directions for permeability data. Fluorescent particle perfusion with electron microscopy identified outflow pathways through the AGs. RESULTS: This human ex vivo model demonstrated in vivo properties of unidirectionality, particle transport, and ultrastructure, similar to our 2005 in vitro model. The average baseline hydraulic conductivity in the physiological direction (n = 20) was 1.05 +/- 0.15 microL/min/mm Hg/cm(2) compared with 0.11 +/- 0.03 microL/min/mm Hg/cm(2) in the nonphysiological direction (n = 3) under statistically equivalent (P = 0.46) average normal physiological pressures (5.88 +/- 0.22 mm Hg and 6.14 +/- 0.23 mm Hg, respectively). CONCLUSIONS: The ex vivo model is feasible and herein demonstrated. These findings agree with in vivo CSF outflow. This model increases understanding of the clearance not only of CSF but also of metabolites through the arachnoid membrane. Additional evidence suggests, but does not yet prove, that CSF outflow may occur in a similar manner in the arachnoid membrane adjacent to the granulations, in addition to the flow through the AGs. This is a topic for further investigation.

Optical quality and impact resistance comparisons of 2 football helmet faceshields.

BACKGROUND: Currently there is no standard that specifically addresses the optical and impact performance of football protective faceshields. This study compared the impact resistance and optical quality between 2 popular football faceshields. Testing was performed only on new faceshields. METHODS: To test impact resistance, baseballs were propelled at the faceshields with velocities up to 66.4 m/sec. Structural integrity was evaluated after each impact. Ten visors from each of 2 companies underwent a single impact at various velocities. Two visors from each company were impacted 3 times to evaluate the effects of repeated blows. Additional visors were conditioned to -10 degrees C and impacted once. Additionally, prismatic power, refractive power, haze, visible light, and ultraviolet (UV) transmittance, and optical distortion were measured to evaluate optical quality. All testing was done with faceshields mounted to facemask and, when appropriate, to a helmet. RESULTS: None of these new faceshields fractured even with impact velocities up to 66.4 m/sec. With regard to optical quality, both protectors met the optical requirements for the standards of faceshields for selected sports (ASTM F803-2003). CONCLUSIONS: Both faceshields tested should protect football players from anticipated impacts while providing adequate optical quality for satisfactory visual performance.

Aggressive immunosuppressive treatment of Susac's syndrome in an adolescent: using treatment of dermatomyositis as a model.

We describe aggressive immunosuppressive treatment of an adolescent with Susac's syndrome (SS), a disease of the microvasculature in the brain, retina, and inner ear. Because the immunopathogenesis of SS appears to have much in common with that of juvenile dermatomyositis (JDM), the patient was treated with an approach that has been effective for severe JDM. The patient's outcome provides evidence for the importance of prompt, aggressive, and sustained immunosuppressive treatment of encephalopathic SS.

Effect of low fluence diode laser irradiation on the hydraulic conductivity of perfused trabecular meshwork endothelial cell monolayers.

OBJECTIVE: To determine the effect of low-fluence diode laser irradiation upon the fluid perfusion characteristics of cultured human trabecular meshwork cell monolayers when placed in a specially designed testing apparatus and subjected to fluid flow driven by a hydrostatic pressure gradient. METHODS: Two experimental series were conducted. In the first series, six low-fluence diode laser irradiation experiments were conducted using cultured human trabecular meshwork cell monolayers grown on filter supports. Upon reaching a steady state perfusion condition at approximately 5.0 mmHg, monolayers were irradiated at fluencies ranging from 0.2619 to 0.8571 J/cm2 using a diode laser (lambda=810 nm). Perfusion and data collection continued for 45 minutes post-irradiation, after which the monolayers were tested to determine post-experimental viability. Hydraulic conductivity values were analyzed for post-irradiation response in 2.5-minute intervals, grouped by viability. In the second series, a total of six irradiated experiments and six simultaneous nonirradiated control experiments were conducted. Fluence values of 0.3571 J/cm2 (n=3) and 0.4286 J/cm2 (n=3) were used. Hydraulic conductivity values were analyzed for post-irradiation response in 2.5-minute intervals, grouped by irradiated vs. nonirradiated control groups. RESULTS: In the first series, analysis showed that the viable monolayers exhibited a statistically significant increase in hydraulic conductivity (p<0.001) from 10 minutes post-irradiation onward. The non-viable monolayers exhibited a statistically significant decrease in hydraulic conductivity. In the second series, irradiated groups showed a significant difference (p<0.001) from nonirradiated controls from 10 minutes post-irradiation onward. CONCLUSION: Low-fluence diode laser irradiation increases hydraulic conductivity in viable perfused TM cell monolayers when compared to baseline values or simultaneous nonirradiated controls while decreasing hydraulic conductivity in nonviable monolayers.

Human arachnoid granulations Part I: a technique for quantifying area and distribution on the superior surface of the cerebral cortex.

BACKGROUND: The arachnoid granulations (AGs) are herniations of the arachnoid membrane into the dural venous sinuses on the surface of the brain. Previous morphological studies of AGs have been limited in scope and only one has mentioned surface area measurements. The purpose of this study was to investigate the topographic distribution of AGs on the superior surface of the cerebral cortex. METHODS: En face images were taken of the superior surface of 35 formalin-fixed human brains. AGs were manually identified using Adobe Photoshop, with a pixel location containing an AG defined as 'positive'. A set of 25 standard fiducial points was marked on each hemisphere for a total of 50 points on each image. The points were connected on each hemisphere to create a segmented image. A standard template was created for each hemisphere by calculating the average position of the 25 fiducial points from all brains. Each segmented image was mapped to the standard template using a linear transformation. A topographic distribution map was produced by calculating the proportion of AG positive images at each pixel in the standard template. The AG surface area was calculated for each hemisphere and for the total brain superior surface. To adjust for different brain sizes, the proportional involvement of AGs was calculated by dividing the AG area by the total area. RESULTS: The total brain average surface area of AGs was 78.53 +/- 13.13 mm2 (n = 35) and average AG proportional involvement was 57.71 x 10(-4) +/- 7.65 x 10(-4). Regression analysis confirmed the reproducibility of AG identification between independent researchers with r2 = 0.97. The surface AGs were localized in the parasagittal planes that coincide with the region of the lateral lacunae. CONCLUSION: The data obtained on the spatial distribution and en face surface area of AGs will be used in an in vitro model of CSF outflow. With an increase in the number of samples, this analysis technique can be used to study the relationship between AG surface area and variables such as age, race and gender.

Characterization of arachnoidal cells cultured on three-dimensional nonwoven PET matrix.

PURPOSE: To culture physiologically functional primary arachnoidal cells on a suitable polymer substrate for an in-vitro model of the cerebrospinal fluid outflow pathway. METHODS: Primary cultures of arachnoidal cells were prepared within 24 hours post-mortem from brain tissue obtained from human cadavers at autopsy. Arachnoidal cells were characterized using immunocytochemistry and seeded onto needle punched non-woven poly(ethylene terephthalate)(PET) scaffolds. Metabolic rate, cell growth rate in log phase, morphologic assessment, immunocytochemistry, and protein analysis were used to characterize the cultures in both 2-D and 3-D-culture. Functional outflow assessment was performed using the Lucifer Yellow (LY) permeability assay and hydraulic conductivity (Lp) determination. RESULTS: Cells cultured on PET scaffold grew slightly slower than cells grown in 2-D-culture as measured by metabolic rate and growth rate, however, they often formed sheets that bridged between the adjacent scaffold filaments forming many junctional protein connections. LY permeability coefficients of 2-D cells were compared with cells from scaffolds, and were not significantly different (p > 0.05) for both culture conditions. Average Lp of cells from 2-D-culture and 3-D-scaffolds were compared and shown not to be significantly different. CONCLUSION: Based on the biochemical and functional analysis, it has been shown that cells cultured on 3D-PET scaffolds retained the same properties as cells from 2D-culture plates.

In vitro model of cerebrospinal fluid outflow through human arachnoid granulations.

PURPOSE: To describe and validate an in vitro model of the arachnoid granulation (AG) outflow pathway for cerebrospinal fluid (CSF), by using human AG cells grown on a filter membrane support and perfused in a modified Ussing chamber at pressures analogous to normal human intracranial pressures. METHODS: Human AG cells were grown, characterized, seeded onto filter membranes, and perfused in the physiologic (basal to apical, B-->A) or nonphysiologic (apical to basal, A-->B) directions. Cells were fixed under pressure after perfusion and prepared for electron microscopy (EM). RESULTS: The average cellular hydraulic conductivity in the B-->A direction (10 total) was 4.52 +/- 0.43 microL/min per mm Hg/cm(2) with an average transcellular pressure decrease of 3.13 +/- 0.09 mm Hg. The average cellular hydraulic conductivity in the A-->B direction (six total) was 0.29 +/- 0.16 microL/min per mm Hg/cm(2) with an average transcellular decrease in pressure of 3.33 +/- 0.16 mm Hg. Cells perfused nonphysiologically showed a large number of dead and dying cells. EM postperfusion analysis showed that AG cells were integrally attached to the underlying filter membrane. Large extracellular cisternal spaces were visible between overlapping AG cells and vacuoles within the cytoplasm. It is possible that these spaces within and between cells represent pathways for transcellular and paracellular transport of fluid. CONCLUSIONS: The results demonstrate that AG cells in vitro show a statistically significant greater flow rate and cellular hydraulic conductivity when perfused in the physiologic versus the nonphysiologic direction under normal intracranial pressures. These results suggest that this in vitro model of the AGs can accurately replicate the unidirectional flow of CSF in vivo.

Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue.

BACKGROUND: The arachnoid granulations (AGs) are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF) to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow. METHODS: Human AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7-10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7-10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144) and their expression was quantified using flow cytometry analysis. RESULTS: Confluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall. CONCLUSION: To our knowledge, this is the first report of the in vitro culture of arachnoidal cells grown from human AG tissue. We demonstrated that these cells in vitro continue to express some of the cytoskeletal and junctional proteins characterized previously in human AG tissue, such as proteins involved in the formation of gap junctions, desmosomes, epithelial specific adherens junctions, as well as tight junctions. These junctional proteins in particular may be important in allowing these arachnoidal cells to regulate CSF outflow.

Model for nonectatic increase in posterior corneal elevation after ablative procedures.

PURPOSE: To evaluate the response of the posterior cornea after uneventful laser in situ keratomileusis (LASIK) with scanning-slit videokeratography using the change in elevation as a measure of biomechanical remodeling. SETTING: Hong Kong Sanatorium and Hospital, Hong Kong, China. METHODS: Three schematic models of possible posterior corneal response mechanisms to laser refractive surgery were proposed: model A, isolated central bulging; model B, forward shift over a large region of the posterior surface; model C, backward peripheral movement into the anterior chamber. A retrospective study was performed to determine which model was consistent with posterior corneal behavior in 2380 eyes of 1255 patients after myopic LASIK performed with the Technolas 217 laser (Bausch & Lomb). Patients received preoperative and 6-month postoperative Orbscan I (version 2.0, Bausch & Lomb) examinations. Preoperative posterior elevation maps were subtracted from postoperative maps of 2380 eyes using 3 fitting protocols. Difference maps were averaged and the results compared to the proposed schematic models. All topography data processing was done using the Ohio State University Corneal Topography Tool. RESULTS: Significant central increases in posterior elevation corresponded with significant peripheral decreases in posterior elevation. This pattern is consistent with schematic model C, backward peripheral movement of the posterior cornea, possibly due to a differential pattern of swelling. CONCLUSIONS: Increases in central posterior corneal elevation after LASIK appear to be dominated by backward peripheral corneal swelling into the anterior chamber rather than forward "bulging" of the central posterior cornea. This is consistent with stable remodeling of the corneal shape due to a surgically induced change in structure and fluid balance, rather than an ectatic event.

Spatial comparison between wall shear stress measures and porcine arterial endothelial permeability.

A better understanding of how hemodynamic factors affect the integrity and function of the vascular endothelium is necessary to appreciate more fully how atherosclerosis is initiated and promoted. A novel technique is presented to assess the relation between fluid dynamic variables and the permeability of the endothelium to macromolecules. Fully anesthetized, domestic swine were intravenously injected with the albumin marker Evans blue dye, which was allowed to circulate for 90 min. After the animals were euthanized, silicone casts were made of the abdominal aorta and its iliac branches. Pulsatile flow calculations were subsequently made in computational regions derived from the casts. The distribution of the calculated time-dependent wall shear stress in the external iliac branches was directly compared on a point-by-point basis with the spatially varying in vivo uptake of Evans blue dye in the same arteries. The results indicate that in vivo endothelial permeability to albumin decreases with increasing time-average shear stress over the normal range. Additionally, endothelial permeability increases slightly with oscillatory shear index.