ABSTRACT
The specific structure of the blood-brain barrier (BBB) is based on the partnership of brain endothelial cells and astrocytes. In the last decade, cocultures of these two cell types have been developed as in vitro models. However, these studies did not allow close contacts between both cell types. We report here a syngenic coculture model using rat endothelial cells on one side of a polyethylene terephtalate filter and rat astrocytes on the other. Endothelial cells retain their typical morphology and are factor VIII and OX 26 positive. We optimized the diameter of the membrane pores to establish very close contacts between the cells through the membrane pores without mixing the two cell types. Transmission electron microscopy showed evidence of tight junction formation between the endothelial cells and few pinocytic vesicles. The cocultures reached high electrical resistances up to 1000 Omegacm(2) showing their ability to limit the passage of ions. A 15-fold increase in gamma-glutamyl transpeptidase activity was measured in the endothelial cells in coculture compared to endothelial cell monoculture. Our syngenic coculture represents a useful in vitro model of the rat BBB that may prove to be valuable for studying the passage of substances across the barrier as well as other aspects of the BBB function.
Subject(s)
Astrocytes/physiology , Blood-Brain Barrier/physiology , Brain/blood supply , Endothelium, Vascular/physiology , Animals , Astrocytes/cytology , Astrocytes/ultrastructure , Brain/cytology , Cells, Cultured , Coculture Techniques/methods , Electric Impedance , Endothelium, Vascular/cytology , Endothelium, Vascular/ultrastructure , Micropore Filters , Microscopy, Electron , Microscopy, Electron, Scanning , Models, Biological , Rats , gamma-Glutamyltransferase/metabolismABSTRACT
We examined changes in membrane properties upon acidification of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes and evaluated their potential to deliver entrapped tracers in cultured macrophages. Membrane permeability was determined by the release of entrapped calcein or hydroxypyrene-1,3,6-trisulfonic acid (HPTS)-p-xylene-bis-pyridinium bromide (DPX); membrane fusion, by measuring the change in size of the liposomes and the dequenching of octadecylrhodamine-B fluorescence; and change in lipid organization, by 31P nuclear magnetic resonance spectroscopy. Measurement of cell-associated fluorescence and confocal microscopy examination were made on cells incubated with liposomes loaded with HPTS or HPTS-DPX. The biophysical studies showed (i) a lipid reorganization from bilayer to hexagonal phase progressing from pH 8.0 to 5.0, (ii) a membrane permeabilization for pH <6.5, (iii) an increase in the mean diameter of liposomes for pH <6.0, and (iv) a mixing of liposome membranes for pH <5.7. The cellular studies showed (i) an uptake of the liposomes that were brought from pH 7.5-7.0 to 6.5-6.0 and (ii) a release of approximately 15% of the endocytosed marker associated with its partial release from the vesicles (diffuse localization). We conclude that the permeabilization and fusion of pH-sensitive liposomes occur as a consequence of a progressive lipid reorganization upon acidification. These changes may develop intracellularly after phagocytosis and allow for the release of the liposome content in endosomes associated with a redistribution in the cytosol.
Subject(s)
Biophysics/methods , Liposomes/chemistry , Liposomes/metabolism , Animals , Arylsulfonates/metabolism , Cells, Cultured , Cholesterol Esters/chemistry , Hydrogen-Ion Concentration , Liposomes/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Magnetic Resonance Spectroscopy , Mice , Particle Size , Permeability , Phosphatidylcholines/chemistry , Phosphatidylethanolamines/chemistryABSTRACT
An impaired vascular response is implicated in the pathogenesis of dialysis-induced hypotension, which is at least partly related to changes in extracorporeal blood temperature (Temp). However, little is known about changes in core Temp and differences in energy balance between standard and cool dialysis. In this study, core Temp and energy transfer between extracorporeal circuit and patient, as well as the blood pressure response, were assessed during dialysis with standard (37.5 degrees C) and cool (35.5 degrees C) Temp of the dialysate. Nine patients (4 men, 5 women; mean age, 69 +/-10 [SD] years) were studied during low- and standard-Temp dialysis, each serving as his or her own control. Bicarbonate dialysis and hemophane membranes were used. Energy transfer was assessed by continuous measurement of Temp in the arterial (Tart) and venous side (Tven) of the extracorporeal system according to the formula: c. rho.Qb*(Tven - Tart)*t, where c = specific thermal capacity (3.64 kJ/kg* degrees C), Qb = extracorporeal blood flow, rho = density of blood (1,052 kg/m3), and t = dialysis time (hours). Core Temp was also measured by Blood Temperature Monitoring (BTM; Fresenius, Bad Homburg, Germany). Core Temp increased during standard-Temp dialysis (36.7 degrees C +/- 0.3 degrees C to 37.2 degrees C +/- 0.2 degrees C; P < 0.05) despite a small negative energy balance (-85 +/- 43 kJ) from the patient to the extracorporeal circuit. During cool dialysis, energy loss was much more pronounced (-286 +/- 73 kJ; P < 0.05). However, mean core Temp remained stable (36.4 degrees C +/- 0.6 degrees C to 36.4 degrees C +/- 0.3 degrees C; P = not significant), and even increased in some patients with a low predialytic core Temp. Both during standard and cool dialysis, the increase in core Temp during dialysis was significantly related to predialytic core Temp (r = 0.88 and r = 0.77; P < 0.05). Systolic blood pressure (RR) decreased to a greater degree during standard-Temp dialysis compared with cool dialysis (43 +/- 21 v 22 +/- 26 mm Hg; P < 0.05), whereas diastolic RR tended to decrease more (15 +/- 10 v 0 +/- 19 mm Hg; P = 0.07). Core Temp increased in all patients during standard-Temp dialysis despite a small net energy transfer from the patient to the extracorporeal system. Concluding, Core Temp remained generally stable during cool dialysis despite significant energy loss from the patient to the extracorporeal circuit, and even increased in some patients with a low predialytic core Temp. The change in core Temp during standard and cool dialysis was significantly related to the predialytic blood Temp of the patient, both during cool- and standard-Temp dialysis. The results suggest that the hemodialysis procedure itself affects core Temp regulation, which may have important consequences for the vascular response during hypovolemia. The removal of heat by the extracorporeal circuit and/or the activation of autoregulatory mechanisms attempting to preserve core Temp might be responsible for the beneficial hemodynamic effects of cool dialysis.
Subject(s)
Energy Transfer , Extracorporeal Circulation/methods , Renal Dialysis/methods , Aged , Blood Pressure , Body Temperature , Female , Hemodynamics/physiology , Humans , Male , TemperatureABSTRACT
Adult rats were treated by intraperitoneal injection of aluminum gluconate for 3 months. Rats were submitted to the radial maze test to determine the influence of chronic aluminum intoxication on cognitive and noncognitive behavioral processes. Both learning abilities (working memory and reference memory) and rapidity (time spent to respond and to master a trial) were analyzed. Aluminum concentration was evaluated in the brain, serum, and liver to assess aluminum body burden. While hippocampus and neocortex showed a significant increase in aluminum concentration, aluminum treatment did never affect the animal's performance during cue learning or when the insert cues were removed. The only behavioral difference observed was a decrease in rapidity: both the total time to finish a trial and the latency to make the first choice were lengthened in aluminum-intoxicated rats.