Isolation and cultivation of porcine astrocytes.

A procedure for the isolation and cultivation of astrocytes from swine is described. More specifically, the donor animals are adolescent minipigs about 3 months in age and 10 kg in weight. About 20 g of cerebral tissue can be isolated from the piglet, yielding enough astrocytes of homogeneous genetics for experimentation after only one passage in culture. The astrocyte isolation procedure includes mechanical and enzymatic digestion of the brain tissue followed by separation of the brain fragments, based on size and density. Astrocytes are further purified from any residual nonastrocytes by differential attachment during the first passage. The resulting culture is purely astrocytes (>98%) based upon their appearance in phase-contrast microscopy and their uniform expression of glial fibrillary acidic protein. More importantly for our purposes, the astrocytes greatly enhanced the functionality of our in vitro blood-brain barrier model when cocultured with porcine brain capillary endothelial cells.

A model for transport studies of the blood-brain barrier.

The blood-brain barrier (BBB) is the cellular structure between the blood flowing through the brain and the parenchymal tissues of the brain. This physiological barrier is formed by the endothelial cells of the capillary walls. It exquisitely regulates the passage of substances into and out of the brain. Astrocytes (astroglial cells) signal the endothelial cells to adopt BBB characteristics. An in vitro BBB model can be very useful for the study of the nutrition, physiology, and pharmacology of the brain. We took advantage of numerous advances made by previous researchers in this field to develop a co-culture BBB model. Capillary endothelial cells and astrocytes are isolated from the brains of miniature swine and grown on permeable membranes suspended between two chambers of media: analogous to the capillary lumen and the interstitium of the brain, respectively. The endothelial cell isolation procedure includes mechanical and enzymatic digestion of the brain tissue followed by separation of the capillary fragments, based on size and density, from other brain cells. Astrocytes are purified from these "other" cells. The endothelial cells of the capillary fragments proliferate in culture flasks and are then seeded onto the upper surface of a polycarbonate semi-permeable membrane suspended between two chambers of fluid. Astrocytes are seeded on the underside of the membranes. Their close proximity enables the astrocytes to communicate with the endothelial cells and encourage their expression of BBB characteristics without disrupting the endothelial cell monolayer. Transport studies across the monolayer can be conducted by introducing test compounds into the media on one side and observing its appearance on the other side. Mechanisms of transport can also be studied.

Longitudinal changes in zinc transport kinetics, metallothionein and zinc transporter expression in a blood-brain barrier model in response to a moderately excessive zinc environment.

A blood-brain barrier (BBB) model composed of porcine brain capillary endothelial cells (BCEC) was exposed to a moderately excessive zinc environment (50 micromol/L Zn) in cell culture, and longitudinal measurements were made of zinc transport kinetics, ZnT-1 (SLC30A1) expression and changes in the protein concentration of metallothionein (MT), ZnT-1, ZnT-2 (SLC30A2) and Zip1 (SLC39A1). Zinc release by cells of the BBB model significantly increased after 12-24 h of exposure, but decreased back to control levels after 48-96 h, as indicated by transport across the BBB from both the ablumenal (brain) and the lumenal (blood) directions. Expression of ZnT-1, the zinc export protein, increased by 169% within 12 h, but was no longer different from controls after 24 h. Likewise, ZnT-1 protein content increased transiently after 12 h of exposure, but returned to control levels by 24 h. Capacity for zinc uptake and retention increased from both the lumenal and the ablumenal directions within 12-24 h of exposure and remained elevated. MT and ZnT-2 were elevated within 12 h and remained elevated throughout the study. Zip1 was unchanged by the treatment. The BBB's response to a moderately high zinc environment was dynamic and involved multiple mechanisms. The initial response was to increase the cells' capacity to sequester zinc with additional MT and to increase zinc export with the ZnT-1 protein. But the longer-term strategy involved increasing ZnT-2 transporters, presumably to sequester zinc into intracellular vesicles as a mechanism to protect the brain and to maintain brain zinc homeostasis.

Brief heat shock affects the permeability and thermotolerance of an in vitro blood-brain barrier model of porcine brain microvascular endothelial cells.

Heat shock was imposed on an in vitro model of the blood-brain barrier (BBB) by submersion into prewarmed growth medium. Transendothelial electrical resistance (TEER) was used to assess the functional integrity of the endothelial barrier. Consequences of the heat shock were highly dependent upon the temperature and duration of exposure. Temperatures below 47 degrees C required more than 30 s of exposure to significantly impair barrier function, but full recovery occurred within 1 h. When the temperature was 50-54 degrees C, an exposure of only 10 s significantly diminished barrier function. Ten seconds of 51 degrees C or 54 degrees C caused a significant loss of barrier function (45% and 80%, respectively). Full recovery from the 51 degrees C shock occurred within 5 min, while recovery from the 54 degrees C shock required more than 10 h. When the temperature was 57 degrees C or greater, a 3-s duration diminished barrier function by 80%. In response to heat shock, the brain microvascular endothelial cells developed thermotolerance and over-compensated in their ability to form a physiological barrier. The BBB models lost more than 60% of barrier function when initially exposed to 53 degrees C for 5 s but lost only 30% of function when exposed to the same treatment 24 h later. The BBB models over-compensated to produce a reinforced barrier with double the original TEER following repeated application of heat treatment (57 degrees C for 3 s). In vivo experiments will require exquisite manipulation of the temperature and duration in order to achieve the desired opening of the BBB in therapeutic applications.

A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier.

A method for the isolation of porcine atrocytes as a simple extension of a previously described procedure for isolation of brain capillary endothelial cells from adolescent pigs [Methods Cell Sci. 17 (1995) 2] is described. The obtained astroglial culture purified through two passages and by the method of the selective detachment was validated by a phase contrast microscopy and through an immunofluorescent assay for the glial fibrillary acidic protein (GFAP). Porcine astrocytes were co-cultivated with porcine brain capillary endothelial cells (PBCEC) for the development of an in vitro blood-brain barrier (BBB) model. The model was visualized by an electron microscopy and showed elevated transendothellial electrical resistance and reduced inulin permeability. To our knowledge, this is the first report for the establishment of a porcine astrocyte/endothelial cell co-culture BBB model, which avoids interspecies and age differences between the two cell types, usually encountered in the other reported co-culture BBB models. Considering the availability of the porcine brain tissue and the close physiological and anatomical relation between the human and pig brain, the porcine astrocyte/endothelial cell co-culture system can serve as a reliable and easily reproducible model for different in vitro BBB studies.

Zinc status influences zinc transport by porcine brain capillary endothelial cells.

Brain capillary endothelial cells (BCEC) were cultured as an in vitro model of the blood-brain barrier (BBB) and manipulated to investigate how the BBB responds to changes in zinc status. BCEC were grown in minimum essential medium (MEM) with 2% fetal bovine serum and 13% platelet-poor horse serum. A moderate zinc deficiency was imposed by growing the cells in medium containing serums that had previously been dialyzed against EDTA to remove endogenous labile zinc. The control treatment was MEM with undialyzed serums (3 micro mol Zn/L); low-Zn was MEM with dialyzed serums (1.5 micro mol Zn/L); Zn-back was MEM with dialyzed serums, plus ZnCl(2) added back (3 micro mol Zn/L); high-Zn was MEM with undialyzed serums, plus ZnCl(2) (50 micro mol Zn/L). Low-Zn treatment increased (P < 0.02) the rate of zinc uptake into BCEC, relative to control and Zn-back; low-Zn treatment also increased (P < 0.05) the rate of zinc transport across the BCEC into the abluminal chamber (analogous to the brain), relative to control and Zn-back. High-Zn decreased (P < 0.02) the rate of zinc transport across BCEC into the brain, while increasing (P < 0.001) the rate of zinc uptake into BCEC, relative to controls. We conclude that BCEC responded to changes in zinc status by altering the rate of zinc transport in a manner consistent with the BBB actively working to sustain brain zinc homeostasis.

Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships.

Flavonoids are a class of secondary plant phenolics with significant antioxidant and chelating properties. In the human diet, they are most concentrated in fruits, vegetables, wines, teas and cocoa. Their cardioprotective effects stem from the ability to inhibit lipid peroxidation, chelate redox-active metals, and attenuate other processes involving reactive oxygen species. Flavonoids occur in foods primarily as glycosides and polymers that are degraded to variable extents in the digestive tract. Although metabolism of these compounds remains elusive, enteric absorption occurs sufficiently to reduce plasma indices of oxidant status. The propensity of a flavonoid to inhibit free-radical mediated events is governed by its chemical structure. Since these compounds are based on the flavan nucleus, the number, positions, and types of substitutions influence radical scavenging and chelating activity. The diversity and multiple mechanisms of flavonoid action, together with the numerous methods of initiation, detection and measurement of oxidative processes in vitro and in vivo offer plausible explanations for existing discrepancies in structure-activity relationships. Despite some inconsistent lines of evidence, several structure-activity relationships are well established in vitro. Multiple hydroxyl groups confer upon the molecule substantial antioxidant, chelating and prooxidant activity. Methoxy groups introduce unfavorable steric effects and increase lipophilicity and membrane partitioning. A double bond and carbonyl function in the heterocycle or polymerization of the nuclear structure increases activity by affording a more stable flavonoid radical through conjugation and electron delocalization. Further investigation of the metabolism of these phytochemicals is justified to extend structure-activity relationships (SAR) to preventive and therapeutic nutritional strategies.