Experimental models for bloodCNSbarrier observations

Animal models

A number of animal models have been successfully established to study cellular and molecular mechanisms of microbial invasion into the brain. Apart from bacterial species used or animals selected as a host, the information obtained from these models is very much dependent on the mode of inoculation. Intranasal, orogastral, intravenous/intracardial, subcutaneous or intraperitoneal inoculation primarily focus on events on "the blood side", e.g., bacterial and host factors that determine the pathogen's fate within the bloodstream and the potential of CNS invasion. In contrast, experimental models using direct inoculation into the CSF rather highlight pathogenetic events on "the brain side". Notwithstanding bypassing the microbial permeation of blood-CNS barriers artificially, these models have the advantage of reliably inducing lethal infections with reproducible bacterial inocula over a predictable time course [59, 60].

These animal models have contributed considerably to the study of pathogen and host factors such as bacterial virulence traits, microbial invasion genes, intracellular signaling cascades and modes of cellular permeation. Furthermore, they have helped in understanding the complications of meningeal inflammation and evaluating potentially useful agents for treatment therapy [61, 62].

An infant rat model has been widely used to mimic human neonatal bacterial meningitis. An important advantage of this model lies in the development of meningitis after bacterial hematogenous spread similar to human newborn meningitis.

The pathogenesis of meningitis has been studied essentially with two major pathogens, Hib [63, 64] or E. coli [65-68] using many different routes of inoculation (nasopharyngeal, orogastric, subcutaneous, intraperitoneal or intracardial). For other purposes an infant rat model with intracisternal inoculation of S. pneumoniae has been used [69, 70]. Other important meningitis models are performed with adult animals by direct systemic or intra-cerebral inoculation mostly in rabbits [71, 72], rats [73] or mice [74].

In recent years, knockout mice with targeted deletion of specific genes have become a powerful tool in investigating the roles of the different adhesins, cytokines, proteases, and oxidants involved in the inflammatory cascade during bacterial meningitis [75].

Cell culture models

To identify and study cellular and molecular mechanisms of microbial permeation of the blood-CNS barriers, it has become important to model the blood-CNS barriers in vitro [76, 77]. Both primary and immortalized cell culture systems have been established. One of the major potential benefits of these in vitro systems in comparison to animal models lies in the possibilities to measure cellular responses to a variety of stimuli without the risk of interference by possible contributions of other cell types such as neuroglia or resident macrophages. Furthermore, no experimental bias is risked by changes in functional and structural characteristics of the blood-CNS barriers.

Blood brain barrier

As outlined above the BBB principally consists of a tight microvascular endothelium, a basal membrane and the pericytic sheath that have to be crossed by bacteria when entering the CNS. The central component of all models is the BMEC. BMECs are usually harvested from brain homog-enates, purified on dextran gradients and cultured alone or together with supporting glial cells. Many mammal BMECs have been used: rat, mouse, dog, dogs, cattle and human [78-84]. Models using peripheral endothelial cell such as human umbilical vein endothelial cells (HUVECs) have also been introduced, but these systemic endothelial cells are likely not appropriate targets for meningitic bacteria [52].

Extending the potential of cell monolayers, several coculture systems have been developed. Bilayer systems consisting of endothelial and epithelial cocultures separated by a porous membrane offer added complexity of multiple layers that might more closely resemble the in vivo situation and allow examination of microbial penetration and associated effects [85].

Multiple studies have indicated that coculturing of BMECs with astro-cytes or neuroglia on opposing sides of a permeable support has mutual benefits as endothelial cells facilitate astrocyte differentiation but, more importantly, astrocytic metabolism contributes to the formation of BBB properties in BMECs (reviewed in [86]). These culture systems were employed in studies on bacterial interactions with cerebral endothelium, e.g., using S. pneumoniae [83, 87] or E. coli [88, 89].

Primary BMEC isolation is laborious, time consuming and the cells are difficult to maintain in native tissue culture and suffer from contamination. In addition, these cells often lose their typical features such as Factor VIII Rag or y-GTP upon subcultivation. Immortalizations and spontaneous transformations have been reported for mouse, rat, cow and human-derived brain endothelial cells.

The best-studied system so far is a human brain microvascular endo-thelial cell line (HBMEC) that has been derived from a brain biopsy of an adult female with epilepsy. The HBMEC were immortalized by transfection with simian virus 40 large-T antigen [90]. This cell line has proven invaluable in multiple experiments on bacterial interaction with the BBB. Many different bacterial species have been examined, e.g. S. agalactiae [91], S. suis [92], S. pneumoniae [83], N. meningitidis [93], Staphylococcus aureus [94], and H. influenzae [95].

In addition to a bovine cell line [90], a porcine counterpart of HBMEC, an immortalized porcine brain microvascular endothelial cell line (PBMEC/ C1-2) has recently been established by lipofection with simian virus 40 small and large T-antigens [96]. It was shown to maintain its morphological and functional characteristics and was used in several investigations with S. suis [49, 97] and Haemophilus parasuis [98].

BMEC cells in vitro as models of the BBB should exhibit substantial properties of cerebral microvascular endothelium. At best they should express tight junction proteins (such as claudins, occludin and ZO-1 and 2) and adherens junction proteins (such as VE-cadherin and p-catenin) spatially separated to morphologically demonstrate features of a polarized monolayer. Functionally, this should translate to a limited permeability to paracellular tracers (e.g., inulin, sucrose, mannitol or dextran) and to ions, resulting in low permeability coefficients and high transendothelial electrical resistance, respectively [99, 100].

Blood-CSF barrier

As mentioned earlier, the tight CP epithelial lining constitutes the structural correlate of the blood-CSF barrier. The establishment of in vitro models of CP epithelial cells has been a challenge for many years. Several preparation methods of primary cells have been established, all based on the initial experiments with rat and cow cells [101, 102]. Subsequently, other working groups have been successful in culturing primary CP epithelial cells including other species: rabbit [103, 104], rat [105-107], cow [102] and swine [108]. However, many primary cultures have been problematic regarding contaminating fibroblasts.

The CP epithelial cells are principally isolated with enzymatic digestion after mechanical pre-treatment and cultured either on flat bottom culture dishes or in permeable filter inserts, where they are able to maintain a hydrostatic pressure difference between apical and basolateral compartment and, thus, are able to establish an effective hydrodynamic barrier.

Just recently our working group has adopted a primary porcine CP cell model [108] for studies of bacterial interactions at the blood-CSF barrier. We were the first to demonstrate a bacteria-CP interaction in vitro using S. suis [109-111] (see also p. 216).

Several CP cell lines have been established from rat [112], mouse [113] or sheep [114] with varying quality regarding typical markers, phenotypes and especially barrier function. Therefore, their impact regarding questions on CNS infections has been limited so far.

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