Deborah M. Grzybowski

The Role of Glia, Mitochondria, and the Immune System in Glaucoma

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

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.

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 Alzheimers 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.

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 Susacs 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 patients outcome provides evidence for the importance of prompt, aggressive, and sustained immunosuppressive treatment of encephalopathic SS.

Ex Vivo Model of Cerebrospinal Fluid Outflow across Human Arachnoid Granulations

PURPOSE The brains 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.

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

BackgroundThe 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.MethodsHuman 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.ResultsConfluent 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.ConclusionTo 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.

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 AMs 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 µl 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 µl 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 µm 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.

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 β 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 β crystallin, a protein marker of LEC differentiation. Inhibition of GJIC via pre-treatment with a synthetic inhibitor, 18-α glycyrrethinic acid (AGA), reduced ATRA-induced increases in Cx43 and GJIC and partially blocked ATRA-induced β 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.

Modeling the Adaptive Permeability Response of Porcine Iliac Arteries to Acute Changes in Mural Shear

AbstractThe hypothesis that much of the uptake of macromolecules by the vascular wall takes place while the endothelial lining is adapting to changes in its hemodynamic environment is being tested by a series of in vivo measurements of the uptake of Evans-blue-dye-labeled albumin by porcine iliac arteries subjected to acute changes in blood flow. The uptake data are interpreted through an ad hoc model of the dynamic permeability response that is proposed to accompany alterations in mural shear.The model is able to correlate, with a single set of parameters, the vascular response to a variety of experimental protocols, including sustained step increases and decreases in shear, and alternations in shear of various periods. The best-fit parameters of the model suggest that the adaptive response to an increase in shear proceeds with a latency of ∼ 1.5 min and a time constant of ∼ 90 min that is substantially shorter than the response to a decrease in shear.

The role of vitamin A and its CSF metabolites in supporting a novel mechanism of idiopathic intracranial hypertension

Background Elevated levels of retinoic acid (RA) may cause dynamic vitamin A metabolic and genetic transcriptional changes that will lead to decreased cellular viability, proliferation, cellular remodeling, adhesion changes and a resultant decrease in permeability, which contributes to elevated CSFP. RA and subsequent formation of retinyl esters, which act as surfactants may cause a toxic response in the arachnoid membrane leading to elevated CSFP.