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- Materials and methods
In vitro blood–brain barrier (BBB) models using primary rat brain microvessel endothelial cells (BMEC) are often hampered by a lack of culture purity and poor barrier properties. To address these problems, the translation inhibitor puromycin was used to purify rat BMEC cultures. BMEC purities of 99.8% were routinely attained using puromycin treatment, and this technique proved to be far superior to other purification methods of similar difficulty. In contrast to cultures without puromycin treatment, purity of puromycin-treated cultures was unaffected by initial seeding density. Next, rat BMEC monolayer transendothelial electrical resistance (TEER) was increased by glucocorticoid treatment with either corticosterone (CORT) or hydrocortisone (HC), and a corresponding decrease in monolayer permeability to small molecules was observed. Importantly, cultures treated with both puromycin and glucocorticoid attained significantly higher TEER values (CORT 168 ± 13 Ω × cm2; HC 218 ± 66 Ω × cm2) than those treated by the glucocorticoid alone (CORT 57 ± 5 Ω × cm2; HC 70 ± 2 Ω × cm2). Glucocorticoid induction resulted in BMEC morphological changes that accompanied the increases in TEER, and BMEC tight junctions exhibited improved integrity as visualized by the localization of tight junction proteins zonula occluden-1, occludin and claudin-5. The combined use of puromycin and glucocorticoid therefore provides an in vitro system that is well suited for molecular level BBB investigations.
The cerebral microvasculature separates the brain interior from the bloodstream and has been termed the blood–brain barrier (BBB) as a result of its impermeable properties. The BBB assists in maintaining brain homeostasis and protects the brain against harmful blood-borne substances. A single layer of brain microvascular endothelial cells (BMEC) is responsible for the limited solute transfer between blood and brain, and these specialized endothelial cells (EC) display distinctive attributes when compared with peripheral endothelium. Low BMEC permeability results from continuous tight junctions between adjoining ECs (Reese and Karnovsky 1967), low levels of pinocytosis and a general lack of fenestrae (Brightman and Reese 1969; Joo 1971).
Because of the impermeable phenotype, the BBB plays major roles in disease pathology and hinders drug delivery efforts. Because of the inherent difficulties in performing molecular level studies of disease pathology in vivo, and the fact that prediction of BBB drug permeability prior to animal studies would be highly advantageous, a representative in vitro model would be of high utility. Unfortunately, when BMECs are cultured in vitro, cellular de-differentiation results in a loss of many of the functions observed in vivo. Much of this change can be ascribed to the removal of BMECs from their local brain microenvironment. Astrocytes, whose foot processes are highly invested in the abluminal surface of capillaries (Kacem et al. 1998), are thought to confer many of the unique BBB properties (Stewart and Wiley 1981; Janzer and Raff 1987). Pericytes and neurons are also intimately associated with BMECs and, although less well studied, appear to play an important role in the differentiation and regulation of the BBB (Hatashita and Hoff 1990; Lee et al. 1999; Petty and Wettstein 2001; Hori et al. 2004). Considerable effort has been focused on regaining in vivo properties in vitro by reconstitution of the microenvironment (Deli and Joo 1996). Examples include the co-culture of BMECs with astrocytes, which has been shown to increase the transendothelial electrical resistance (TEER) and tight junction complexity, while reducing the paracellular passage of molecules such as sucrose (Tao-Cheng et al. 1987; Giese et al. 1995; Mertsch et al. 1997). The addition of neurons to cultures containing BMECs caused an increase in the enzymatic activities of γ-glutamyl transpeptidase and Na+ K+ ATPase, and induced the correct localization of the tight-junction protein occludin by these cells (Tontsch and Bauer 1991; Savettieri et al. 2000). The role of pericytes has been less well characterized, but in vitro studies indicate that they may have an ability to induce BBB properties (Hori et al. 2004; Dohgu et al. 2005). Additional studies highlight the importance of fluid flow (Stanness et al. 1996), and soluble factors such as cAMP (Rubin et al. 1991; Deli et al. 1995), hydrocortisone (HC) (Hoheisel et al. 1998; Weidenfeller et al. 2005) and dexamethasone (Grabb and Gilbert 1995), that have been shown to assist in partial restoration of in vivo characteristics such as barrier tightness. Although substantial progress has been made to date, in vitro models of the BBB continue to be labor-intensive and difficult to reproduce. In addition, in vitro BBB models tend to exhibit reduced permeability characteristics, a significant shortcoming for studying molecular or cellular trafficking across the BBB.
Many of the problems encountered in developing in vitro models with in vivo permeability characteristics stem from the difficulty of obtaining pure endothelial cultures. While the basement membrane surrounding the microvascular endothelium facilitates the isolation of these structures from total brain material (Panula et al. 1978), it also encloses pericytes that are difficult to remove from the endothelial fraction. As a result, these cells often contaminate BMEC cultures in an uncontrollable manner and have been shown to prevent complete barrier formation (Parkinson and Hacking 2005). Previous research indicates that pericytes in contact with microvascular endothelial cells produce an active form of transforming growth factor type-β which inhibits endothelial growth (Orlidge and D'Amore 1987; Antonelli-Orlidge et al. 1989). Therefore, pericytes often become the predominant cell type in such cultures, increasing the permeability of the BMEC monolayer (Parkinson and Hacking 2005; Perriere et al. 2005). The need for methods that can achieve pure BMEC cultures in a simple and reproducible manner still remains.
Techniques for achieving pure BMEC cultures continue to appear in the literature and these studies demonstrate a correlation between EC purity and improved barrier properties (Parkinson and Hacking 2005; Perriere et al. 2005). Methods for the isolation of microvessel fragments usually include mechanical and/or enzymatic dissociation of the brain material followed by filtration and/or density centrifugation steps (Bowman et al. 1981; Gordon et al. 1991; Deli et al. 2003). Other methods have incorporated magnetic bead separation (Song and Pachter 2003; Parkinson and Hacking 2005) and fluorescence-activated cell sorting after labeling with an endothelial specific marker (Sahagun et al. 1989) to increase endothelial cell purity. As described above, pericytes often constitute a major contaminating cell type in BMEC cultures, but smooth muscle cells, astrocytes, and fibroblasts can also be found (Greenwood 1992; Perriere et al. 2005). Techniques for minimizing contaminating cell types in cultured BMECs include physical separation (Abbott et al. 1992), selective toxicity (Risau et al. 1990), and the appropriate control of serum and growth supplement concentrations (Bowman et al. 1982; Carson and Haudenschild 1986; Gordon et al. 1991; Abbott et al. 1992). Another method of comparative ease employs chemical mediators for achieving culture purity. BMECs express p-glycoprotein (p-gp), an efflux transporter that recognizes a variety of small molecule substrates, some of which are cytotoxic (Litman et al. 2001). BMECs can therefore survive relatively high concentrations of molecules that would be toxic to many cell types. This strategy has been exploited by including vincristine, a p-gp substrate, in the culture medium to purify murine and porcine BMEC cultures (Chen et al. 1998; Igarashi et al. 1999), and puromycin was especially well suited for purifying rat BMEC cultures (Perriere et al. 2005).
We have also demonstrated that puromycin treatment of rat BMEC cultures routinely yields nearly 100% pure EC cultures. The method clearly outperformed other facile purification methods and was reproducible regardless of initial BMEC plating density. To further the impermeability characteristics of these cultures, glucocorticoids were added to BMEC monolayers. While corticosterone is the major physiological glucocorticoid found in rodents, hydrocortisone is a more potent glucocorticoid with respect to its anti-inflammatory and glycogen-deposition effects (Haynes and Murad 1985). Both corticosterone and hydrocortisone yielded improvements in BMEC impermeability, and pure cultures responded much more favorably to glucocorticoid treatment than untreated cultures. Taken together, this study demonstrates the utility of straightforward chemical treatments for significantly improving the permeability characteristics of a highly reproducible in vitro BBB model.
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- Materials and methods
This study has focused on the development of a facile, reproducible method for cultivating rat BMECs as in vitro BBB models having improved permeability properties. To this end, we identified puromycin treatment as being the most robust method for purifying rat BMEC cultures. This method is insensitive to seeding density effects and routinely generates 99.8% EC cultures. Importantly, puromycin-purified cultures respond much more favorably to glucocorticoid induction than do untreated cultures. The impermeability as measured by TEER and fluorescein permeability is greatly improved and is a consequence of well-developed tight junctions.
Puromycin is an antibiotic produced by Streptomyces alboniger that inhibits the growth of Gram-positive bacteria and various animal and insect cells. This aminonucleoside antibiotic functions by specifically inhibiting peptidyl transfer in prokaryotic and eukaryotic ribosomes. BMECs can withstand a relatively high concentration of this molecule because of their expression of the efflux transporter p-gp (MDR1), which recognizes puromycin and effluxes it from these cells. Recently Perriere et al. (2005) demonstrated that puromycin was not toxic to endothelial cells at the concentrations used here, and that chronic treatment could induce mdr1b expression. Inclusion of puromycin in the medium of rat BMEC cultures for the first 2 DIV dramatically increased culture purity, as also reported by this previous study (Fig. 2). Perriere et al. (2005) also demonstrated that puromycin treatment yielded the purest rat BMEC (98.8% pure) monolayers with the least toxicity compared with other p-gp substrates, vincristine, vinblastine, colchicine, and doxorubicin. Similarly, we have shown that puromycin yields 99.8% pure BMEC cultures. In our hands, this treatment was the vastly superior method for attaining culture purity.
Another technique compared in the purity analysis was the use of Ca2+- and Mg2+-free saline washes of cultures after 1.5 and 2.5 DIV, the period during which contaminating cell outgrowth became significant. The brief treatment of cultures with Ca2+- and Mg2+-free saline has been reported to preferentially remove contaminating cell types leaving the ECs behind (Abbott et al. 1992). This technique promoted little increase in culture purity (Fig. 2). Pre-plating of freshly isolated capillary fragments onto uncoated culture surfaces was also attempted in an effort to remove contaminating cell types. The basis of this method is rooted in the observation that ECs have been shown to adhere and spread well to collagen and fibronectin but poorly to uncoated plastic or glass (Gordon et al. 1991; Abbott et al. 1992). As pericytes and smooth muscle cells adhere relatively well to uncoated plastic, the strategy involves pre-plating the cell solution onto an uncoated plastic surface for several hours (Gordon et al. 1991; Dore-Duffy 2003). The pre-plating step removes the faster adhering cells from the ECs, which are then seeded onto a coated culture surface. Similar to the conclusions found in Parkinson and Hacking (2005), this method was unsuccessful despite these previous reports (Gordon et al. 1991; Dore-Duffy 2003). The failure of this method to increase purity was likely because of the loss of cellular material as a result of the pre-plating step, resulting in a very low relative seeding density and therefore a higher contaminating cell fraction after 5 DIV (see below). Thus, these cultures did not attain confluency and the method lacked reproducibility.
Besides the use of puromycin, a major key to achieving high-purity BMEC cultures was the replacement of FBS with PDS as a source of serum. By switching the FBS medium supplement to PDS in cultures lacking puromycin treatment, purities of near 84% were obtained, representing a 15% increase in purity. This increase can be attributed to the lack of platelet-derived growth factor in PDS, which reduces the growth rate of pericytes and smooth muscle cells while having little effect on the EC growth rate (Bowman et al. 1982; Carson and Haudenschild 1986; Gordon et al. 1991; Abbott et al. 1992). The use of PDS and puromycin treatment provided for culture purities routinely as high as 99.8%, while cultures grown in FBS with puromycin treatment were not quite as pure (97.4%). The lower purity of FBS-containing cultures was mainly because of the presence of a small population of cells expressing both von Willebrand factor and α-actin that were therefore not categorized as ECs. Morphologically, these cells appeared to be of endothelial origin, as evidenced by their spindle shape and their tight packing into the monolayer. Indeed, there is previous evidence of porcine brain ECs reversibly expressing α-actin in culture (Amberger et al. 1991). Therefore, the use of FBS may either promote the growth of a subpopulation of ECs that express α-actin or induce the expression of α-actin in a subpopulation of ECs. Either way, an EC expressing α-actin represents a de-differentiated form, so the use of PDS was preferred to assist in maintaining BMEC phenotype.
In order to test the experimental robustness of the puromycin method, cultures were seeded at varying initial cell densities. Cultures treated with puromycin were essentially pure regardless of the initial seeding density (Fig. 3). Thus, the use of puromycin provides a high degree of reproducibility between experiments by consistently attaining high purity despite the inevitable differences between microvessel preparations. In contrast, in untreated cultures, purity was found to be a function of initial seeding density with a very small window of seeding densities achieving maximal (> 85%) purity and confluence (Fig. 3). In low seeding density cultures where a confluent monolayer of BMECs was not formed by 5 DIV (< 1.2 × 105 cells/cm2), contaminating cell types grew on the substrate surface in addition to growing on top of existing BMEC colonies. The production of active transforming growth factor-β by such co-cultures can inhibit EC growth (Orlidge and D'Amore 1987; Antonelli-Orlidge et al. 1989) and prevent the formation of confluent monolayers. Accordingly, for cultures where high purity and yield are needed, the use of puromycin and PDS is preferred, as a broad range of initial cell seeding densities can result in confluent monolayers.
TEER is a general measure of paracellular ion flux, and serves as a proxy measurement for assessing EC barrier function (Hoheisel et al. 1998). When primary rat BMECs were cultured on Transwell-Clear® filters with or without puromycin, TEER measurements revealed a statistically significant increase in TEER values for cultures having puromycin treatment (Fig. 4). The study by Perriere et al. (2005) also reported an increase in TEER for puromycin-treated cultures as compared with untreated cultures. This can likely be attributed to the decrease in the number of pericytes and other contaminating cells, a notion supported by the recent findings of Parkinson and Hacking (2005), who showed that pericyte reduction caused a decrease in monolayer permeability to sucrose.
In order to further improve barrier properties, serum-free medium containing CORT or HC in the range of physiological concentrations was added to BMEC monolayers and resulted in significantly increased TEER (Fig. 4) [1400 nm for corticosterone (Karlson et al. 1994), 550 nm for hydrocortisone (Vahl et al. 2005)]. To our knowledge, this is the first demonstration of using the predominant rodent glucocorticoid, corticosterone, to improve in vitro BBB barrier properties. Interestingly, glucocorticoid addition to puromycin-treated samples resulted in greater TEER values than those seen without puromycin treatment. These results indicate that puromycin-treated monolayers respond more favorably to glucocorticoid treatment than untreated monolayers, and demonstrate that the use of both puromycin and glucocorticoids can allow for the reproducible formation of ‘in vivo-like’ BBB models. The most impermeable rat BBB models typically yield TEER values in the same range as those reported here (218 ± 66 Ω × cm2 for HC; de Vries et al. 1996; Demeuse et al. 2002), while in vitro models using BMEC from other species can at times yield higher TEER [bovine 600 Ω × cm2 (Zysk et al. 2001), and porcine 1800 Ω × cm2 (Nitz et al. 2003)]. After the 24-h time period, the effects of serum-free culture begin to lessen the benefits of glucocorticoid induction, and the TEER drops (Fig. 4). However, serum-free conditions were used in this study as the addition of glucocorticoids to serum-containing medium (10% serum) results only in minimal BBB induction (Hoheisel et al. 1998).
The BBB models described here responded to glucocorticoid treatment on a reliable timescale and maintained their maximal barrier properties for hours, enabling their use in permeability studies. As an example, the functional permeability of monolayers was determined using the normally BBB-impermeant small molecule, fluorescein. Mapping with elevations in TEER, puromycin treatment significantly lowered monolayer permeability, while HC induction further decreased the permeability (Table 1). The fluorescein permeability values determined for the puromycin (8.3 × 10−6 cm/s) and HC-treated cultures (1.1 × 10−6 cm/s) compare favorably with the lowest fluorescein permeability values reported for rat BMEC–astrocyte co-culture BBB models (0.75–4.2 × 10−6 cm/s; Kis et al. 2001; Perriere et al. 2005). The fluorescein permeability for the puromycin and HC system also compares well with that observed for bovine (2.2 × 10−6 cm/s; Gaillard and de Boer 2000) or human BBB models (53 × 10−6 cm/s; Muruganandam et al. 2002). In addition to soluble mediators like CORT and HC, puromycin-treated BMECs have also been shown to respond to astrocyte induction (Perriere et al. 2005), raising the possibility that puromycin-treated BMECs can be used in studies focused on intercellular interactions that are prevalent at the BBB.
The postulated physical basis for the observed improvements in barrier properties after puromycin and glucocorticoid treatment includes morphological changes and alterations in junctional structure. First, an immunocytochemical analysis revealed that more complete monolayers formed in cultures treated with puromycin, and that these cultures were populated with far fewer pericytes than in untreated cultures (Fig. 4, time 0; Table 1). Parkinson and Hacking (2005) described a similar finding noting the superior barrier properties of BMEC cultures with fewer pericytes. Next, upon glucocorticoid induction, a switch in EC morphology from a larger, slightly cobblestone appearance to a smaller, spindle shape took place leading to a monolayer having increased EC density (Table 1). Proliferation assays suggested that the increased cell density was not attributable to an increase in the proliferation rate as a consequence of HC treatment, but instead was because of a decrease in the rate of cell detachment in HC-treated cultures. This result is consistent with a previous study illustrating the anti-apoptotic effects of hydrocortisone in a porcine in vitro BBB model (Arndt et al. 2004). It is of note that, despite the higher putative cell detachment rate of puromycin-treated cultures as compared with cultures treated with both puromycin and HC, cultures lacking HC were still capable of maintaining confluent monolayers (Table 1).
The changes in morphology were accompanied by higher TEER values, lower permeability coefficients, and a less frayed appearance along the cell borders, as evidenced by staining for ZO-1, occludin and claudin-5 tight junction proteins (Table 1; Fig. 5). It is also interesting to note that gross changes in the cellular actin distribution did not accompany the observed refinement in junctional structure. The association between frayed tight-junction structure and TEER was previously noted by Weidenfeller et al. (2005). Their study employed transmission electron microscopy to demonstrate an HC-mediated morphological switch from frayed to continuous tight junctions, and this switch corresponded to increases in TEER. Taken together with our observations, these studies indicate that glucocorticoid treatment results in tighter packing (reduced cellular area) and fewer frayed junctions that serve to promote higher TEER values.
Recent evidence suggested that HC increases occludin mRNA and protein levels in an immortalized mouse brain endothelial cell line by activating the glucocorticoid receptor and its binding to putative glucocorticoid responsive elements in the occludin promoter (Forster et al. 2005). In addition, ZO-1 expression was induced in an immortalized rat brain endothelial cell line (GPNT) upon dexamethasone addition (Romero et al. 2003). In contrast to these systems, the study described here employed non-transformed primary endothelial cell cultures, and the net per cell expression of occludin and claudin-5 did not change following hydrocortisone treatment. Although net expression per cell does not change, it is still possible that tight junction protein turnover may be increased during HC-induced junction rearrangements and that the synthesis of new tight junction proteins may be up-regulated to assist in remodeling cell–cell junctions.
Methods for isolating brain microvessels invariably result in the co-isolation of some level of contaminating cells. Our results validate the use of puromycin for the reproducible attainment of essentially pure rat BMEC cultures. These purified cells respond optimally to glucocorticoid induction by the formation of a tighter barrier, as evidenced by increased TEER and reduced fluorescein permeability, without the use of additional cell types or conditioned media. While not all applications require a pure EC system, our model would be well suited for several experimental goals, including drug permeability screening and leukocyte trafficking. Furthermore, as a consequence of the BMEC purity, this model may be extremely helpful in ascribing observed attributes specifically to endothelial cells and not to cellular culture contaminants. This benefit may be particularly important for genomic and proteomic analyses focused on deconvoluting the complex roles that different cell types or soluble factors have in conferring BBB properties.