Purification and characterization of metabolically active capillaries of the blood-brain barrier

Abstract

Microvessels were isolated from bovine and rat cerebral cortex by simple procedures involving mechanical homogenization, differential and density-gradient centrifugation, and chromatography on a column of glass beads. The preparations were composed of short capillaries with a diameter of 1-10 microns. Both purifications were monitored by assaying the activity of the marker enzyme gamma-glutamyl transpeptidase (gamma-GTase). The final bovine and rat preparations were enriched 20- and 14-fold over the homogenate respectively. gamma-GTase activity was measured in different fractions after bovine and rat membranes were solubilized with 0.5% and 0.3% Triton X-100 respectively. Measurement of 5'-nucleotidase and acetylcholinesterase activities indicated very low levels of contamination of the microvessel preparations by glial cells and neurons. The integrity of the capillary membranes was confirmed by the assay of a cytosolic marker enzyme, lactate dehydrogenase. Viability of the microvessels was demonstrated by the presence of detectable levels of adenylates and by tissue respiration induced by glucose and succinate. Comparison of the proteins of homogenized bovine and rat brain cortex with those of purified capillaries separated by SDS/PAGE revealed enrichment of at least three predominant proteins of 14, 16 and 18 kDa in the capillary preparations. It is concluded that these methods allow rapid isolation of small blood vessels of the blood-brain barrier which are suitable for metabolic and structural studies in vitro.

Negative Relationship Between Morphine Analgesia and P-Glycoprotein Expression Levels in the Brain

“…The protein concentration was determined with the Coomassie (Bradford) Protein Assay Kit (Pierce Chemical), with bovine serum albumin as the standard. The membrane fraction obtained by this method is known to be highly enriched for the marker enzymes gamma-glutamyl transpeptidase and alkaline phosphatases (17). We confirmed the expression of P-gp in our membrane preparation of BCECs using western blot analysis (data not shown).…”

“…All steps in the isolation procedure were carried out at 4°C. In regard to this method, the prepared brain capillary fraction has been morphologically characterized by means of phase-contrast microscopy (17). The capillary preparation was resuspended in 1 ml of 0.1% (w / v) collagenase II in phosphate-buffered saline (PBS) and incubated at 37°C for 30 min.…”

“…Mouse brain capillaries were isolated from mouse cerebrum, as described previously with some modifications (16,17). Briefly, individual mouse brain cortices (for one experiment, mixed brains of 2 to 4 mice were used) were stripped of the pial membrane, meninges, superficial large blood vessels, and chroid plexus and then gently homogenized in 3 volumes (v / w) of ice-cold physiological buffer (PB), consisting of 147 mM NaCl, 4 mM KCl, 3 mM CaCl 2 , 1.2 mM MgCl 2 , 5 mM glucose, and 15 mM HEPES, pH 7.4, with a tissue homogenizer run at 400 rpm for 20 strokes.…”

Inhibition of Src Activity Decreases Tyrosine Phosphorylation of Occludin in Brain Capillaries and Attenuates Increase in Permeability of the Blood-Brain Barrier after Transient Focal Cerebral Ischemia

“…Isolation of brain capillaries was carried out using a modified version of the method described earlier (Dallaire et al, 1991). Rats at 1, 6, 12, and 24 h of reperfusion were killed by decapitation, and their heads were quickly nearfrozen in liquid nitrogen.…”

High transcytosis of melanotransferrin (P97) across the blood–brain barrier

“…P97 accumulation in human brain capillaries Human brain capillaries were isolated by a procedure previously described (Dallaire et al 1991;Demeule et al 2001). A rapid filtration technique was used to measure the accumulation of of 5 lM of unlabeled P97, holo-transferrin or lactoferrin.…”