Lymphatic cells do not functionally integrate into 3D organotypic brain slice cultures, but aggregate around penetrating blood vessels.

Brain slice culture (BSC) is a well-known three-dimensional model of the brain. In this study, we use organotypic slices for studying neuro-lymphatic physiology, to directly test the longstanding assumption that the brain is not a hospitable milieu for typical lymphatic vessels. An additional objective is to model fluid egress through brain perivascular space systems and to visualize potential cellular interactions among cells in the leptomeninges including alterations of cellular geometry and number of processes. Immortalized lymphatic rat cell lines were used to seed organotypic brain slices. The brain slice model was characterized by monitoring morphologies, growth rates, degree of apoptosis, and transport properties of brain slices with or without a lymphatic component. The model was then challenged with fibroblast co-cultures, as a control cell that is not normally found in the brain. Immortalized lymphatic cells penetrated the brain slices within 2-4 days. Typical cell morphology is spindly with bipolar and tripolar forms well represented. Significantly more indigo carmine marker passed through lymphatic seeded BSCs compared to arachnoid BSCs. Significantly more indigo carmine passed through brain slices co-cultured with fibroblast compared to lymphatic and arachnoid BSCs alone. We have developed an organotypic model in which lymphatic cells are able to interact with parenchymal cells in the cerebrum. Their presence appears to alter the small molecule transport ability of whole-brain slices. Lymphatic cells decreased dye transport in BSCs, possibly by altering the perivascular space. Given their direct contact with the CSF, they may affect convectional and diffusional processes. Our model shows that a decrease in lymphatic cell growth may reduce the brain slice's transport capabilities.

Cell growth of immortalized arachnoid cells in the presence of fibroblasts and blood products.

OBJECT: The pathophysiology of non-obstructive hydrocephalus involves alteration in cerebrospinal fluid (CSF) pathways. The exact mechanism is unknown, but as arachnoid CSF egress is a major route of CSF removal, damage or alteration to the growth of arachnoid cells may influence the rate of CSF absorption. We investigated the effect of soluble factors secreted by fibroblasts and the presence of blood products on arachnoid cell growth. METHODS: An immortalized arachnoid cell line was developed and cells were grown on semipermeable membranes in a culture chamber. Arachnoid cells were plated in Transwells®, with fibroblasts separated from the arachnoid cells. Cell phenotype was analyzed and cell growth rates were determined by manual counts. Similar experiments were conducted with biliverdin, bilirubin, as well as fibroblast challenge. DNA content in the cell cultures was then determined as corroborative data. Cell counts for the additional arachnoid cell lines were calculated at each day and represented the controls. RESULTS: Cell counts increased with each time point. Arachnoid cells in the three experimental conditions showed a statistically significant decrease in cell counts for each day when compared to the control group. Post hoc analysis showed differences between the control and experimental conditions but no significant difference between groups. The DNA content for each experimental condition was reduced at all time points when compared to the control arachnoid cells, but only became statistically significant at day 7. CONCLUSION: Inflammation and hemorrhage are two common conditions associated with the development of hydrocephalus. The arachnoid membrane is exposed to fibroblasts and blood products (bilirubin, biliverdin) in these conditions, and their effect on arachnoid cell growth was studied. We have shown that arachnoid cell growth decreases in the presence of fibroblasts, bilirubin, and biliverdin. Given its intimate relationship with CSF, it is possible that the decreased growth of arachnoid cells may affect absorption and thus contribute to the development of hydrocephalus.

The effects of blood and blood products on the arachnoid cell.

After traumatic brain injury (TBI), large amounts of red blood cells and hemolytic products are deposited intracranially creating debris in the cerebrospinal fluid (CSF). This debris, which includes heme and bilirubin, is cleared via the arachnoid granulations and lymphatic systems. However, the mechanisms by which erythrocytes and their breakdown products interfere with normal CSF dynamics remain poorly defined. The purpose of this study was to model in vitro how blood breakdown products affect arachnoid cells at the CSF-blood barrier, and the extent to which the resorption of CSF into the venous drainage system is mechanically impaired following TBI. Arachnoid cells were grown to confluency on permeable membranes. Rates of growth and apoptosis were measured in the presence of blood and lysed blood, changes in transepithelial electrical resistance (TEER) was measured in the presence of blood and hemoglobin, and small molecule permeability was determined in the presence of blood, lysed blood, bilirubin, and biliverdin. These results were directly compared with an established rat brain endothelial cell line (RBEC4) co-cultured with rat brain astrocytes. We found that arachnoid cells grown in the presence of whole or lysed erythrocytes had significantly slower growth rates than controls. Bilirubin and biliverdin, despite their low solubilities, altered the paracellular transport of arachnoid cells more than the acute blood breakdown components of whole and lysed blood. Mannitol permeability was up to four times higher in biliverdin treatments than controls, and arachnoid membranes demonstrated significantly decreased small molecule permeabilities in the presence of whole and lysed blood. We conclude that short-term (<24 h) arachnoid cell transport and long-term (>5 days) arachnoid cell viability are affected by blood and blood breakdown products, with important consequences for CSF flow and blood clearance after TBI.

The influence of fibroblast on the arachnoid leptomeningeal cells in vitro.

OBJECTIVE: Fibroblast is pervasive in the setting of injury. Its invasion into the arachnoid tissue causes scarring, cortical adhesion of the brain, and obstruction of cerebrospinal fluid outflow. The purpose of this study is to determine the phenotypic and physiologic effects of fibroblasts on arachnoid in culture. METHODS: We studied the effects of fibroblast on the arachnoid cell growth, motility, phenotypic changes, and transport properties. Immortalized rat (Rattus norvegicus, Sprague Dawley breed) arachnoid cells were grown with fibroblast on opposite sides of polyethylene membranes or co-cultured in plastic wells. Arachnoid cell growth rate and DNA content, morphology, transport physiology, and extracellular matriceal content were determined in the presence of normal and irradiated fibroblast cells. RESULTS: When arachnoid cells were grown in the presence of fibroblasts, mannitol permeability increased and transepithelial electrical resistance (TEER) decreased. Arachnoid cell growth rate also significantly decreased. When arachnoid cells were grown in close proximity (i.e. on the same monolayer) with fibroblasts, the arachnoid cells were overrun by day 2, yet when physically separated, no significant change was seen in growth. Apoptosis increased markedly in arachnoid cultures in the presence of fibroblast. Fibroblast caused arachnoid cell to exhibit avoidance behavior, and irradiated fibroblast induced arachnoidal cells to move faster and exhibited greater directional changes. Subcellular glycosaminoglycan (GAG) content was significantly altered by fibroblast. INTERPRETATION: Fibroblasts influence arachnoid cell's mannitol transport likely via soluble factors. While the arachnoid cells did not change morphologically, cell growth was influenced. Over time, the cells had profound changes in transport and motility. The immortalized arachnoid cell/fibroblast culture system provides a unique model mimicking the pathologic event of leptomeningeal scarring.

Generation and preliminary characterization of immortalized cell line derived from rat lymphatic capillaries.

OBJECTIVE: Isolation of rodent endothelial cells from lymphatic capillaries with yields that allow extensive functional studies remains challenging due to low cell numbers, variable purity, and limited growth potential. The purpose of this study was the generation and preliminary characterization of rat lymphatic cell line with extended replicative capacity. This cell line is intended for in vitro studies of cellular transport in lymphatic endothelium and for in vivo experiments in rat animal models. METHODS: We created a novel rat lymphatic immortalized cell line, SV40-LEC, using retroviral gene transfer of SV40 large T antigen. We confirmed expression of characteristic markers and then examined its growth and transport properties. RESULTS: SV40-LECs demonstrated improved proliferative capacity, but retained morphological characteristics of lymphatic cells and expression of established lymphatic markers. The cells form capillary-like network in vitro. SV40-LEC monolayer has similar permeability to that of the primary initial lymphatics. Paracellular transport in SV40-LECs is limited for substances >70 kDa. Barrier properties of the SV40-LECs can be modulated by cyclic adenosine monophosphate and histamine, which are known to affect microvascular permeability. CONCLUSION: The SV40-LECs provide an excellent tool for in vitro studies of properties of lymphatic endothelium, and may be suitable for in vivo transplantation studies.

Arachnoid cells on culture plates and collagen scaffolds: phenotype and transport properties.

INTRODUCTION: The arachnoid tissue is a critical component of cerebrospinal fluid removal. Failure of that function results in hydrocephalus, a serious medical condition. The purpose of this study was to characterize arachnoid cell transport in culture and on three-dimensional collagen scaffold. METHODS: Arachnoid cells were harvested from rat brainstems and cultured onto bilayered bovine collagen scaffolds. Cell growth and phenotype (protein expression and morphometry) were determined. Permeability and hydraulic conductivity were quantified. RESULTS: Cells harvested from the anterior brainstem surface exhibited arachnoid cell phenotype (positive for vimentin, desmoplakin, and cytokeratin), readily penetrated the collagen scaffold, and doubled approximately every 2-3 days. The transepithelial electrical resistance value for a monolayer of cells was 160 Ω cm(2) and the permeability of indigo carmine was 6.7×10(-6)±1.1×10(-6) cm/s. Hydraulic conductivity of the collagen construct was 6.39 mL/min/mmHg/cm(2). CONCLUSION: Cells isolated from the anterior brain stem exhibited the same phenotype as those found in the native tissue and exhibited aspects of barrier function found in vivo. These studies suggest that an ex vivo model for the arachnoid granulation can be developed.