Transmural gradients of oxygen tension on cerebral microvessels in rats

Using modified oxygen needle microelectrodes, vital microscopy with video-recording facilities, measurements of tissue oxygen tension (PO2) profiles near the cortical arterioles and transmural PO2 gradients on pial arterioles of the rat were performed. At control transmural PO2 gradient averaged 1.17 +/- 0.06 mm Hg/microm (mean +/- SEM, n = 40). Local dilatation of the arteriolar wall (microapplication of sodium nitroprusside approximately 2 x 10(-7) M) resulted in marked drop of the transmural PO2 gradient to 0.68 +/- 0.04 mm Hg/microm (p < 0.001, n = 38). The important finding of the study is the dependence of the transmural PO2 gradient on the vascular tone of pial arterioles. The data presented allow to conclude that O2 consumption of the arteriolar wall lies within the range for surrounding tissue and O2 consumption of the endothelial layer and, apparently, has no substantial impact on transmural PO2 gradient.     

Red blood cell velocity and oxygen tension measurement in cerebral microvessels by double-wavelength photoexcitation.

Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.     

Impaired dilation of skeletal muscle microvessels to reduced oxygen tension in diabetic obese Zucker rats

This study determined alterations to hypoxic dilation of isolated skeletal muscle resistance arteries (gracilis arteries; viewed via television microscopy) from obese Zucker rats (OZR) compared with lean Zucker rats (LZR). Hypoxic dilation was reduced in OZR compared with LZR. Endothelium removal and cyclooxygenase inhibition (indomethacin) severely reduced this response in both groups, although nitric oxide synthase inhibition (N(omega)-nitro-L-arginine methyl ester) reduced dilation in LZR only. Treatment of vessels with a PGH(2)-thromboxane A(2) receptor antagonist had no effect on hypoxic dilation in either group. Arterial dilation to arachidonic acid, iloprost, acetylcholine, and sodium nitroprusside was reduced in OZR versus LZR, although dilation to forskolin and aprikalim was unaltered. Treatment of arteries from OZR with oxidative radical scavengers increased dilation to hypoxia and agonists, with no effect on responses in LZR. The restored hypoxic dilation in OZR was abolished by indomethacin. These results suggest that hypoxic dilation of skeletal muscle microvessels from LZR represents the summated effects of prostanoid and nitric oxide release, whereas the impaired response of vessels in OZR may reflect scavenging of PGI(2) by superoxide anion.     

Cell-free plasma layer in cerebral microvessels.

Two diameters of vessel and red cell column in cerebral microvessels (> 29.8 microns in diameter) of cat were measured together with red cell velocity, using a two fluorescent tracer method. A fluorescein isothiocyanate (FITC)-labeled red cell was adopted as a flow tracer to measure the cell velocity with a dual window technique. Based on the fluorescence image, the red cell column diameter was measured. Plasma was stained with rhodamine-B isothiocyanate (RITC)-labeled dextran to measure the vessel diameter. The thickness of the cell-free plasma layer could be determined from the difference of the two diameters. The obtained thickness of the cell-free layer was not described by a simple function of vessel diameter or red cell velocity; it was dependent on the pseudo shear rate defined by the ratio of cell velocity to vessel radius. The layer thickness increased with a decrease in the pseudo shear rate.     

Longitudinal oxygen gradients in cerebra micro vessels in acute anaemia in rats

Using a fine-tip oxygen microelectrodes the longitudinal gradients of oxygen tension (pO2) have been studied in small arterioles (with lumen diameter in control of 5 +/- 20 microm) and in capillaries of the rat brain cortex during stepwise decrease of the blood haemoglobin concentration [Hb] from control [Hb]--14.4 +/- 0.3 g/dl to 10.1 +/- 0.2 g/dl (step 1), 7.0 +/- 0.2 g/dl (step 2) and 3.7 +/- 0.2 g/dl (step 3). All data are presented as "mean +/- standard error". Oxygen tension was measured in arteriolar segments in two locations distanced deltaL = 265 +/- 34 microm, n = 30. Mean diameter of studied arterioles was 10.7 +/- 0.5 microm, n = 71. Length of studied capillary segments was about deltaL = 201 +/- 45 Mm, n = 18. The measured longitudinal pO2 gradient (deltapO2/deltaL) in arterioles amounted 0.03 +/- 0.01 mmHg/microm, n = 15 in control; 0.06 +/- 0.01 mmHg/microm, n = 16 (step 1); 0.07 +/- +/- 0.01 mmHg/microm, n = 14 (step 2); 0.1 +/- 0.01 mmHg/microm, n = 30 (step 3). In the capillaries, the deltapO2/deltaL amounted to: 0.07 +/- 0.01 mmHg/microm, n = 17 (control); 0.09 +/- 0.02 mmHg/microm, n = 16 (step 1); 0.08 +/- 0.01 mmHg/microm, n = 15 (step 2); 0.1 +/- 0.02 mmHg/microm, n = 18 (step 3). An over threefold decrease in the system blood oxygen capacity did not result in significant changes (p > 0.05) of the deltapO2/deltaL in capillaries that might result in relatively homogeneous oxygen flux from blood to tissue in acute anaemia. The longitudinal gradients of blood O2 saturation (deltaSO2/deltaL) in studied arterioles and capillaries were obtained using oxygen dissociation curve (ODC) of haemoglobin in the system blood. The gradients deltaSO2/deltaL in capillaries was shown to be threefold higher than the corresponding gradients in arterioles. The data show that anatomic capillaries are the main source of oxygen to brain tissue as in control and in hypoxic conditions. Sufficient oxygen delivery to brain tissue in acute anaemia is maintained by compensatory mechanisms of cardiovascular and respiratory systems. The data presented are the first measurements of the longitudinal pO, gradients in capillaries and minute cortical arterioles at acute anaemia.     

Membrane-bound lipoxygenase of rat cerebral microvessels

The microvessels isolated from rat cerebral cortex has arachidonate lipoxygenase activity, which was not due to possible contamination of the platelets. The major product was identified to be 12-hydroxyeicosatetraenoic acid. After homogenization and sonication of the microvessel preparations, the lipoxygenase activity was recovered both in the membrane- and the cytosol-fractions, whereas that in the platelets was recovered in the cytosol fraction. Membrane-bound lipoxygenase of the microvessels has apparent Km value of 3.8 microM for arachidonic acid, which was corresponded to 1/5 of that in the platelet enzyme. Microvessel lipoxygenase was inhibited by nordihydroguaiaretic acid but not by indomethacin.     

Oxygen tension in thein vivo cornea

The steady state distribution of oxygen tension in thein vivo cornea is estimated in the present study by using a nonlinear oxygen consumption rate equation of the Michaelis-Menten type. Such a rate expression is more accurate than the previous simplified versions in predicting the oxygen consumption rate. It is found that for an open eye with or without contact lens, the oxygen tensions predicted previously are in good agreement with these predicted in the present work. However, for a closed eye with or without contact lens, the previous predictions underestimate the oxygen tension.     

Protection against radical peroxidations in cerebral aging in cerebral capillaries and microvessels]

Systemic injection of substances that generate free radicals into the rat induces deleterious alteration of various tissues (as demonstrated by the production of conjugated dienes and malondialdehyde) but only has a slight effect on the brain. This shows that the blood-brain barrier has important protective properties. In fact the cerebral capillaries and microvessels have superoxide dismutase, glutathione peroxidase, and catalase activities higher than those found in the rest of the cerebral tissue during aging. These activities vary little, except for catalase which decreases. But, curiously, the concentrations of Mn, Cu, and Zn are not related to enzymatic activity, although the micro-elements are necessary for the activity of superoxide dismutase. On the other hand, during aging, the capillaries and cerebral microvessels undergo extensive modifications at the level of the polyunsaturated fatty acids: for example, the concentration of arachidonic acid decreases by half.     

Expression of P-glycoprotein in human cerebral cortex microvessels.

P-Glycoprotein (P-gp) is an ATP-dependent efflux transporter that extrudes non-polar molecules, including cytotoxic substances and drugs, from the cells. It was initially found in cancer cells and then was shown to be a normal component of complex transport systems working at the blood-brain barrier (BBB). Previous studies have demonstrated that, in the brain, P-gp is localized on the luminal plasmalemma of BBB endothelial cells and that it may interact with the caveolar compartment of these cells. The aim of this study was to identify the site of cellular expression of P-gp in human brain in situ and to morphologically determine whether an association may exist between P-gp and caveolin-1, a structural and functional protein of the caveolar frame. The study was carried out on human cerebral cortex by immunoconfocal microscopy with antibodies to both P-gp and caveolin-1. The results show that P-gp marks the microvessels of the cortex and that the transporter is localized in the luminal endothelial compartment, where it co-localizes with caveolin-1. The demonstration of this co-localization of P-gp with caveolin-1 contributes a morphological backing to biochemical studies on P-gp/caveolin-1 relationships and leads us to suggest that interactions between these molecules may occur at the BBB endothelia.     

Parathyroid hormone stimulates adenylate cyclase in rat cerebral microvessels

We have studied the effect of parathyroid hormone (PTH) on adenylate cyclase of microvessels isolated from rat cerebral cortex. Native bovine (b) PTH-(1–84), the synthetic amino-terminal fragment bPTH-(1–34) and the synthetic analog [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH- (1–34) amide stimulated adenylate cyclase in a dose-dependent manner with apparent ED₅₀ values of 16 nM, 6.3 nM and 15 nM respectively. The stimulation by bPTH was greatly enhanced by guanosine triphosphate. The PTH antagonist, [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH-(3–34) amide inhibited the action of bPTH-(1–84) and bPTH-(1–34). In summary, PTH stimulated adenylate cyclase in rat cerebral microvessels in a very similar manner to its stimulation in the renal cortex.     

beta-Adrenergic receptors in brain microvessels of diabetic rats

A significant decrease in the number of beta-adrenergic receptors was observed in cerebral microvessels of fatty (fa/fa) and streptozotocin-induced diabetic rats, without receptor affinity changes. These results suggest that alterations of central adrenergic regulation of small vessels may be involved in brain microvasculature disturbances that occur with diabetes.     

Effect of oxygen tension on chromosomal aberrations in Fanconi anaemia

Blood samples from four healthy individuals and from seven Fanconi anaemia (FA) patients were cultured at oxygen tensions ranging from 3% to 45% O2. Cultures were harvested at 72 h and scored for chromosomal aberrations. In the majority of FA patients the aberration frequency showed a tendency to increase as a function of oxygen tension over the culture, whereas the aberration frequency in healthy individuals was not affected. However, the response in FA cultures was variable among patients and in individual cases when assayed on different occasions. A much stronger effect of oxygen tension was observed when the FA blood samples had been treated with mitomycin C (0.25 microgram/ml, 30 min) before culture initiation.     

Prostaglandin D synthase in microvessels from the cat cerebral cortex

Microvessels, a mixture composed predominantly of small arterioles and capillaries (7–80μ diameter), were isolated from the rat cerebral cortex by selective nylon sieving and glass bead elutriation. The morphology and purity of the microvessel and cerebral cortex filtrate (virtually free of vascular contamination) were monitored by light microscopy and by the activity of several enzymes: γ-glutamyl transpeptidase, GSH-transferase, prostacyclin synthase and PGD synthase. Prostacyclin and PGD synthesizing activities as well as γ-glutamyl transpeptidase activity were localized to the microvessels of the rat cerebral cortex whereas GSH-S-transferase was restricted to the non-vascular filtrate function. The characteristics of the PGD synthase were similar to those of the purified enzyme previously described for the rat brain. The microvessel (MV) PGD synthase was localized to the cytosol fraction of the microvessels and did not require reduced glutathione for activity. The enzyme was inhibitd by pre-incubation with p-hydroxymercuribenzoate (lmM) or N-ethylmaleimide (lmM). The MV PGD synthase saturated at 15–20μM PGH₂, exhibited an apparent K_M of 9.6μM, and a pH optimum of 8.0–8.1. These findings suggest roles for both prostacyclin and PGD synthesis by the rat cerebral vasculature in the autoregulation of cerebral blood flow and/or function. These studies also indicate that the major source of PGI₂ and PGD₂ synthesis by rat brain homogenates is the microvasculature.     

Diabetes-related changes in the protein composition of rat cerebral microvessels

To determine the effect of diabetes mellitus on cerebral microvessel protein composition, post translational modification of proteins with glucose and malondialdehyde (MDA) was determined and the abundant protein species found in cerebral microvessels isolated from control and streptozotocin-induced diabetic rats were studied. Two dimensional gel electrophoresis and computer assisted densitometry revealed that only one out of 25 quantitated proteins was significantly altered in diabetic rats after 5 weeks of uncontrolled hyperglycemia. The level of glycosylation of cerebral microvessel protein mixture was significantly increased in diabetic rats compared to control rats (168.8±25 vs 109.5±4.8 nmol/mg) (p<0.05). Western blot analysis of cerebral microvessel proteins from diabetic rats using a specific antibody against MDA-modified proteins revealed three protein spots with molecular weights of approximately 60,000 Kd. These were shown not to be contaminants from cerebral tissue or plasma proteins modified with MDA. It is concluded that short duration of streptozotocin-induced diabetes mellitus in rats is associated with some qualitative changes in protein composition of cerebral microvessels. These changes may contribute to the diabetes-related alterations in the blood-brain barrier.