Human Arachnoid Granulation Topography

A CSF Outflow Ex Vivo Model

Future Study to Include Topological Examination, Search for Aquaporins

A human CSF outflow ex vivo model has also been created for the first time at Ohio State. Another member of the Gryzbowski/Katz research team and the lead author on this study, Shelley Glimcher, B.S., presented her work on the creation of the model, as well as ideas for future research involving the model.

Instead of using only cap cells in perfusion studies, the ex vivo model incorporates the entire arachnoid granulation structure. The role of whole tissue perfusion of the human arachnoid membrane—in other words, how CSF flows through this structure—was key in this study since it is a major component of the blood-CSF barrier and can provide insight into the mechanism that triggers idiopathic intracranial hypertension. No previous work has been done on whole tissue perfusion of human arachnoid granulations.

Ms. Glimcher noted that by measuring CSF outflow through the arachnoid granulations and micro villi simultaneously, the researchers will be able to observe interaction among all cell types, thereby gaining a better understanding of the physiology. This idea forms the basis for the ex vivomodel.

She also described how a perfusion apparatus was constructed in the lab. Donated human arachnoid granulation tissue (less than 12 hours post-mortem)
samples were cut to size and secured in a special chamber, where they were oriented for fluid to flow from basal to apical. The tissue was perfused for 16-18 hours, with pressure across the membrane in preliminary experiments ranging from 4.0 to 5.5 mm. Hg. After five perfusion runs, enough data was collected to determine that a viable ex vivo model of CSF outflow through the human arachnoid membrane had been created.

Further trials will help determine correct concentrations of tracers, which can be used to determine pore size distribution. Analysis of stained tissue sections from the arachnoid membrane will also expand current knowledge about the arachnoid granulations and the effects of CSF outflow. Plans for future study include the topological examination of different sections of the arachnoid granulations; elevated pressure perfusions runs; the use of microparticles to determine where and how much of the fluid is perfusing across the membranes; and the staining of tissue for the proteins which control water movement (aquaporins).

A CSF Outflow In Vitro Model

Team achieves IH research milestone

Pia-arachnoid cells, also known as “cap cells,”are located at the top of the arachnoid granulations and are thought to control the unidirectional CSF flow through the arachnoid granulations. In order to better understand this process, the Gryzbowski/Katz research team at Ohio State focused on growing cap cells in their lab to create the first CSF outflow in vitro model.

David Holman, M.S., a member of the Gryzbowski/ Katz research team and the lead author on this particular study, explained how cap cells were grown. Using donated brain tissue collected at autopsy, samples were transferred to cell culture plates, where cell growth occurred in seven to ten days. The newly
grown cap cells were checked for certain proteins to determine if the cultured cells identically mimicked cap cells found in humans.

The cultured cap cells were also grown on a special filter membrane designed to measure hydraulic conductivity. The cells were perfused at a fixed pressure and their hydraulic conductivity was successfully measured, indicating a successful in vitro model, as well. Future studies of the CSF outflow in vitro model’s hydraulic conductivity will test a variety of different conditions including increased fluid pressure and the effect of Vitamin A.

New Directions in CSF Outflow Research

A research roadmap was presented by Deborah Grzybowski, Ph.D, Assistant Professor of Ophthalmology and Biomedical Engineering at the Ohio State University in Columbus, OH and a course director for the Neural Hydrodynamics Symposium. The research is being jointly lead by Dr. Steven Katz, Associate Professor of Ophthalmology at Ohio State, an IHRF Scientific Advisor and a fellow course director for the NHD Symposium.

There is limited knowledge about CSF physiology, especially regarding the areas and mechanisms of fl uid movement from the subarachnoid space. Dr. Gryzbowski and Dr. Katz hypothesize that an increase in intracranial pressure is caused by increased resistance at the pia-arachnoid cell layer in the arachnoid granulations, the tiny channels that drain CSF into the cerebral blood circulation. In other words, CSF outflow resistance in the pia-arachnoid cells could be what causes intracranial hypertension.

To determine whether this theory is true, Drs. Gryzbowski and Katz have undertaken a multi-faceted approach to identify proteins and receptors
that might influence CSF outfl ow. They proposed both clinical and basic science research. Clinical projects, in collaboration with the IH Registry, include a genetic study of IIH patients, who have at least two family members with the disorder.

A second project will analyze levels of Vitamin A and its analogues, transport proteins, specific receptors, and obesity regulation proteins and hormones in both CSF and blood serum of IIH patients. A third clinical project will study fluid movement of pre- and post-shunt IIH patients, using a diffusion tensor MRI scan to measure whether brain edema and increased diff usion are factors in IIH.

Drs. Gryzbowski and Katz have already accomplished one of their basic science goals by successfully growing human pia-arachnoid cells in their lab for the first time in history. This achievement will significantly clarify the role of these cells in CSF outflow.

Additionally, basic science projects include brain mapping of the arachnoid granulations and villi; studying the role of Vitamin A and its eff ect on CSF outflow, as well as identifying specific proteins and receptors (carbonic anhydrase, somatostatin, alpha-adrenergic, beta-adrenergic and the Vitamin A transduction pathway) that infl uence outfl ow; and the development of ex vivo and in vitro CSF outflow models.

The development of an IH animal model is also among their priorities.

The Lymphatic System and CSF Drainage

An Alternate Route to IH Etiology?

Miles Johnston, Ph.D., Professor of Laboratory Medicine and Pathobiology at the University of Toronto in Toronto, Canada, offered a different perspective on CSF drainage and the lymphatic system. The commonly-held belief maintains that CSF drains through the tiny channels or villi of the arachnoid granulations into the venous blood system.

But a link between CSF and the lymphatic system has been known for over a century. Dr. Johnston suggested that the lymphatic system may act as a drainage conduit for CSF, based on animal models and anatomical evidence. He discovered that in many diff erent animals, increasing intracranial pressure
elevates lymphatic fl ow in the neck. Whether the same is true in humans is unknown.

Additionally, in animals, if CSF access to paranasal lymphatics is blocked, cranial CSF absorption is reduced. In a sheep model, some CSF drainage may
have occurred directly into the cranial venous system (blood circulation), though Dr. Johnston found that there was little evidence that this absorption took place through the arachnoid granulations. According to the study, the lymphatic anomalies may be associated with disorders of the
CSF system. Hydrocephalus animal models have revealed physical abnormalities in the lymphatic system.

Dr. Johnston suggested that lymph may be a main or secondary pathway for draining CSF. He also theorized that elevated intracranial pressure may be the result of an impaired lymphatic system that cannot drain CSF properly, which would mean that improving lymphatic drainage would lower intracranial pressure.

Na-K-Cl co-transporter and its role in CSF formation

Na-K-Cl Co-Transporter, Neuropeptides, and Choroid Epithelial ‘Neuroendocrine’ Cells Impact CSF Production

The Symposium’s keynote speaker, Conrad Johanson, Ph.D., Professor of Clinical Neuroscience and the Director of Neurosurgical Research at the Brown Medical School in Providence, RI, as well as an IHRF Scientific Advisor, presented his work on the Na-K-Cl co-transporter and its role in CSF formation and ion homeostasis.

Dr. Johanson found that the Na-K-Cl transporter can bi-directionally transport sodium (Na), potassium (K) and chloride (Cl) ions into and out of CSF. These ions must be kept in homeostatic balance. It is likely that the Na-K-Cl transporter also plays an important part in modulating CSF production and perhaps, under certain circumstances, reabsorption. Neuropeptides also help to regulate CSF formation. Th ey can alter ion transport and cause neuroendocrine-like “dark cells” to develop in the epithelial layer of the choroid plexus, the site where CSF is produced in the brain. (“Dark cells” appear when there is CSF inhibition.)

According to Dr. Johanson’s research, several changes occur in a hydrocephalus model including an increase in CSF; neuroendocrine receptor plasticity in the choroid plexus; an increase in the width of the intercellular space between epithelial cells of the choroid plexus (suggesting CSF inhibition); an increase in the number of “dark cells”; and an increase in the expression of the Na-K-Cl cotransporter. These changes are thought to be signs of a decrease in CSF production in order to re-establish homeostasis (a process known as downregulating).

Downregulating is believed to occur in response to elevated intracranial pressure. If this holds true, then these findings could be a major discovery as to whether
intracranial hypertension is caused by an impediment to CSF outflow or by CSF hypersecretion. The evidence from Dr. Johanson’s study does not support the theory of CSF hypersecretion.

The Field of Neural Hydrodynamics

Ghassan Bejjani, M.D., Clinical Assistant Professor of Neurosurgery at the University of Pittsburgh Medical Center and an IHRF Scientific Advisor, opened the Symposium with a discussion of the similarities between disorders of increased intracranial pressure.

Citing an example from a paper that he authored, Dr. Bejjani reported significant similarities between adult Chiari malformation (ACM) and idiopathic intracranial hypertension (IIH) including patient demographics, presenting symptoms and response to treatment. He also discovered that there was an eight-fold increase in the incidence of significant tonsillar herniation in the IIH patients studied, and suggested that disorders of increased intracranial pressure—including ACM and IIH—are interrelated.According to his paper, one clue to the close connection between ACM and IIH may be a physical disproportion between the size of the skull and the size of the brain.

Dr. Bejjani stressed the importance of focusing on the common links between intracranial hypertension, hydrocephalus, Chiari malformation and syringomyelia, as well as developing common common treatments for patients with these disorders.

Choroid Plexus-CSF Homeostatic Systems

Disruption of Choroid Plexus-CSF Homeostatic Systems in Aging and Alzheimer’s Disease

IHRF Scientific Advisor and this year’s NHD Symposium keynote speaker Conrad Johanson, Ph.D. brought the research conference to a close with a discussion about the consequences of a decrease in CSF production with aging. Dr. Johanson believes that reduced CSF formation leads to a disruption in CSF flow dynamics and reduced CSF turnover, which, in turn, allows for damaging toxins, like beta amyloid, to accrue in the brain.

CSF formation in humans, as well as animals, decreases by 50% or more in the late stages of life. This occurs as a result of the diminishing expression of Na-K-ATPase, carbonic anhydrase and aquaporins in the choroid epithelium.
A breakdown in the choroid plexus secretory systems also occurs with aging. Choroidal fluid generation is critical to extracellular fluid balance in the central nervous system, and is necessary to maintain CSF volume, pressure and chemical composition. Actual examination of the choroid plexus has shown physical changes including calcifi cation, immune complex deposition and interstitial fibrosis, all of which lead to less eff ective transfer of solutes across the blood-CSF barrier.

In addition, Dr. Johanson reported that lower CSF turnover that occurs with aging means that essential nutrients are not supplied efficiently to neurons while potentially harmful, plaque-like substances known as catabolites are slowly and
ineffectually removed. (Beta amyloid is a catabolite that has been associated with Alzheimer’s disease.)

This aging-induced disruption in CSF dynamics likely contributes to the exacerbation of dementias such as Alzheimer’s disease (AD). Dr. Johanson discovered that in severe AD, certain proteins involved in homeostatic regulation had been upregulated. The fluid regulator NA-K-Cl co-transporter in the apical membrane of the choroid plexus was also found at a higher expression level.

Dr. Johanson suggested that enhanced reabsorptive activity of the Na-K-Cl cotransporter is associated with the slowing of CSF production. The development of drugs to increase or regulate CSF formation may help to solve the problems associated with altered CSF flow dynamics.