Electrolyte- and fluid-spaces of rat brain in situ after infusion with dinitrophenol

Chemical distribution measurements of radioactive sodium-thiosulfate (³⁵S) and of the brain water indicate that infusion of 2.4-dinitrophenol into a carotid artery of rats caused a water uptake and fluid shifts from the extra- into the intracellular compartments in the central nervous system. The extracellular marker compound was administered to the brain via ventriculo-cisternal perfusion and intravenous injection yielding almost equal concentrations in plasma- water and perfusate. In order to prevent an active efflux of the label from the tissue, high concentrations were utilized in the perfusate to saturate potential outward transport mechanisms. The indicator space (based on total brain water) was 16% in controls and 12% in experimental animals when marker equilibrium had been attained, which is equivalent in reduction of the extracellular space of about 1/4. Intracellular water and Na⁺ rose after DNP, while K⁺ remained all but unchanged. The fluid shift into the intracellular compartment was found to relate closely with a cellular uptake of Na⁺. The Na⁺ concentration both in plasma and in the perfusion fluid leaving the ventricular system was consistently reduced in experimental animals. The K⁺ concentration was significantly elevated in the plasma of experimental animals but virtually unchanged in the cisternal effluate.     

Heme-copper relationship of cytochrome oxidase in rat brain in situ

The role of copper aa3 in relation to heme aa3 of cytochrome oxidase in electron transfer and oxygen utilization is poorly understood in vitro. In an attempt to study this in situ, we have simultaneously monitored the steady state redox changes of heme aa3 and copper aa3 in an isolated perfused rat head model (skull intact, muscle removed). By means of reflectance spectrophotometry the redox reactions of heme aa3 and copper aa3 were continuously monitored using 605-625 nm and 815-920 nm wavelength pairs, respectively. The reaction kinetics of these components in response to transient perfusion interruption in energized and de-energized preparations were then examined. We found that in response to perfusion interruption, soon after full reduction, the copper signal begins to change toward oxidation despite continuation of anoxic insult and progressive reduction of heme aa3. This phenomenon disappeared by pretreatment of the preparation with 2,4-dinitrophenol. A schematic sequence of electron transport in situ is proposed which emphasizes an active role for Cua in this sequence.     

In situ localization of P-glycoprotein (ABCB1) in human and rat brain.

Transport of several xenobiotics including pharmacological agents into or out of the central nervous system (CNS) involves the expression of ATP-dependent, membrane-bound efflux transport proteins such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Previous studies have documented gene and protein expression of P-gp in brain microvessel endothelial cells. However, the exact localization of P-gp, particularly at the abluminal side of the BBB, remains controversial. In the present study we examined the cellular/subcellular distribution of P-gp in situ in rat and human brain tissues using immunogold cytochemistry at the electron microscope level. P-gp localizes to both the luminal and abluminal membranes of capillary endothelial cells as well as to adjacent pericytes and astrocytes. Subcellularly, P-gp is distributed along the nuclear envelope, in caveolae, cytoplasmic vesicles, Golgi complex, and rough endoplasmic reticulum (RER). These results provide evidence for the expression of P-gp in human and rodent brain capillary along their plasma membranes as well as at sites of protein synthesis, glycosylation, and membrane trafficking. In addition, its presence at the luminal and abluminal poles of the BBB, including pericytes and astrocyte plasma membranes, suggests that this glycoprotein may regulate drug transport processes in the entire CNS BBB at both the cellular and subcellular level.     

Anabolic sialosylation of gangliosides in situ in rat brain cortical slices

Radiolabeling of the sialic acid residues of gangliosides was examined in thin slices of rat brain cerebral cortex incubated under physiologic conditions in the presence of either [14C]N-acetyl-mannosamine (ManNAc) or cytidine 5'-monophosphoryl-[14C]N-acetyl-neuraminic acid (CMP-NeuAc). CMP-NeuAc is the direct donor substrate in the transfer of sialic acid to gangliosides by sialosyl transferases (SATs), including ectosialosyl transferases at the cell surface. ManNAc must be internalized by the neural cells (neuronal or glial) where it serves as an obligate precursor for the biosynthesis of the NeuAc moiety of intracellular CMP-NeuAc, via multiple reactions in the cytosol and nucleus. When exogenous [14C]ManNAc was supplied, there appeared to be a 2-h lag period before label was incorporated measurably into ganglioside sialic acid. That was followed by rapid ganglioside labeling continuing up to 6 h. There was high incorporation into ganglioside GM1. Labeling by ManNAc was inhibited by monensin, a monovalent cationophore that blocks anabolic transport in medial and trans Golgi. Extracellular CMP-NeuAc was not internalized by the cells. CMP-[14C]NeuAc labeling of gangliosides had no lag period, reached a maximum within 2 h, and then began to level. The label distribution among gangliosides was high in GD3, but quite low in GM1. CMP-NeuAc labeling was not inhibited by 10(-7) M monensin. These findings support a model in which ManNAc labels gangliosides by an intracellular route involving monensin-sensitive, Golgi-associated SATs. In this intracellular system, the major labeled products are gangliosides of the gangliotetraosyl series (GM1, GD1a, etc.).(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution in situ hybridization and TUNEL staining with free-floating brain sections.

We applied in situ hybridization and the TUNEL technique to free-floating (vibratomed) sections of embryonic and postnatal mouse CNS. Full-length cDNAs specific for oligodendrocyte- or astrocyte-specific genes were labeled with digoxigenin using the random primer method. With paraformaldehyde-fixed sections, the nonradioactive in situ hybridization method provides detection of individual, very small glial progenitor cells in embryonic development. Small, isolated cells expressing oligodendrocyte specific messages can be detected in the neuroepithelium at embryonic and postnatal stages. The technique can be completed within 3 days and is as sensitive as the radioactive method. Likewise, the TUNEL method using DAB as the chromogen on free-floating sections provides excellent resolution. These DAB-stained sections can be embedded in plastic and thin-sectioned to visualize the ultrastructure of apoptotic cells. Both in situ hybridization and TUNEL methods can be applied to the same section, the tissue embedded in plastic, and semithin sections cut. The high resolution obtained with this combined procedure makes it possible to determine whether brain cells expressing glia-specific messages are undergoing apoptosis.     

Effect of lactate infusion on M-wave characteristics and force in the rat plantaris muscle during repeated stimulation in situ.

It is unclear whether accumulation of lactate in skeletal muscle during exercise contributes to muscle fatigue. The purpose of the present study was to examine the effect of lactate infusion on muscle fatigue during prolonged indirect stimulation in situ. For this purpose, the plantaris muscle was electrically stimulated (50 Hz, for 200 ms, every 2.7 s, 5 V) in situ through the sciatic nerve to perform concentric contractions for 60 min while either saline or lactate was infused intravenously (8 rats/group). Lactate infusion (lactate concentration approximately 12 mM) attenuated the reduction in submaximal dynamic force (-49 vs. -68% in rats infused with saline; P < 0.05). Maximum dynamic and isometric forces at the end of the period of stimulation were also higher (P < 0.05) in rats infused with lactate (3.8 +/- 0.3 and 4.4 +/- 0.3 N) compared with saline (3.1 +/- 0.2 and 3.6 +/- 0.2 N). The beneficial effect of lactate infusion on muscle force during prolonged stimulation was associated with a better maintenance of M-wave characteristics compared with control. In contrast, lactate infusion was not associated with any reduction in muscle glycogen utilization or with any reduction of fatigue at the neuromuscular junction (as assessed through maximal direct muscle stimulation: 200 Hz, 200 ms, 150 V).     

Activation of the phosphorothionate insecticide parathion by rat brain in situ

The ability of the rat brain to activate the phosphorothionate insecticide parathion to its potent anticholinesterase metabolite paraoxon in situ was observed by ligating the posterior portion of the circulatory system and thus removing the liver from the circulation. Under these conditions no acetylcholinesterase inhibition was observed in 15 min at a dosage of parathion (nominally 2.4 mg/kg) which yielded 95% inhibition when the liver was in the circulation. However, at a higher dose (nominally 48 mg/kg) there was substantial (about 70%) inhibition of brain acetylcholinesterase after 15 min, suggesting that the brain does have the ability to activate parathion in the intact situation.     

Accumulation of labeled gamma-aminobutyric acid into rat brain and brain synaptosomes after i.p. injection

The accumulation of labeled GABA into brain and brain nerve endings was studied in the adult rat after i.p. injection of large doses of neurotransmitter (740 mg/Kg). In the first 5–30 minutes after the injection the exogenous neurotransmitter reaches a stable plasma level of around 5 mM. The accumulation of radioactive GABA into the brain presents a latency of a few minutes from the time of the injection. Thereafter, the accumulation of the neurotransmitter is almost linear with time. Once in the brain tissue labeled GABA is in part broken down. The exogenous neurotransmitter is taken up in GABA-ergic nerve endings with a steep increase between 20 and 30 minutes after the injection. From a quantitative point of view, the data show that the brain accumulation of labeled GABA at 30 minutes post injection is minimal in the respect of the steady state average concentration of the endogenous neurotransmitter (0.014%). However, the amount of radioactive GABA which accumulates in the nerve endings, at the same post injection time, is around 7% of the endogenous neurotransmitter in that comparment. The data thus show a selective enrichment of exogenous systemic GABA in a physiologically important compartment of the brain.     

Different skeletal regional response to continuous brain infusion of leptin in the rat

This study was designed to evaluate whether or not continuous intracerebroventricular infusion of leptin (1.5 microg/rat/24 h, for 28 days) produced different regional response on the skeleton of growing rats. Leptin reduce the accretion of total femoral bone mineral content (BMC) and density (BMD). This effect was related to a reduction of metaphyseal femur as no changes were detected in the diaphysis. Despite the reduced accretion in the volumetric of both femur and tibia compared to controls, leptin had no significant effects on the lumbar vertebrae. Urine deoxypyrydinoline and serum osteocalcin remained more elevated in the leptin-treated group as compared to controls. The results demonstrate that long-term central infusion of leptin activates bone remodeling with a negative balance. Leptin induces distinct responses in the different structure of bone and in the axial and appendicular skeleton.     

Changes in fatty acid composition in rat blood and organs after infusion of eicosapentaenoic acid ethyl ester.

An infusible emulsion of 10% eicosapentaenoic acid ethyl ester (EPA-EE, 99.2% pure) was prepared. Three ml of the emulsion was infused into tail veins of 20 Wistar rats weighing approx. 300 g. They were killed 1 h, 6 h, 24 h, 3 d and 7 d after the infusion, and fatty acid composition of various organs and plasma was analyzed along with that of control rats. There was no lipidosis in any organs of any rats. It was estimated that not less than 98% of infused EPA was cleared from plasma during the first hour after the infusion. EPA concentrations reached their peaks within 6 h after EPA infusion in plasma lipid fractions and in the phospholipid fraction of liver, heart, lung, kidney and spleen. The fatty acid composition of the phospholipid fraction of heart was very stable, and was not altered by EPA infusion except for a very slight increase in EPA at 1 h after the infusion (0.18% at 0 h to 0.56%). However, EPA concentrations increased markedly in the free fatty acid fraction of heart at 1 h after the infusion (0.15% at 0 h to 4.23%). Arachidonic acid concentrations decreased significantly within 24 h in the phospholipid fraction of organs except for heart. EPA emulsion might be useful for patients in whom a rapid increment in EPA in tissues is beneficial.