Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats.
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Hippocampal slices have long been used to investigate properties of synaptic transmission and plasticity. Here, for the first time, synapses in slices have been compared quantitatively with synapses occurring in perfusion-fixed hippocampus, which is presumed to represent the natural in vivo state. Relative to perfusion-fixed hippocampus, a remarkable 40-50% increase in spine number occurs in adult hippocampal slices, and a 90% increase occurs in slices from postnatal day 21 rats. Serial EM shows that all of the dendritic spines have normal synapses with presynaptic and postsynaptic elements; however, not all spine types are affected uniformly. Stubby and mushroom spines increase in the adult slices, and thin, mushroom, and branched spines increase in the immature slices. More axonal boutons with multiple synapses occur in the slices, suggesting that the new synapses form on preexisting axonal boutons. The increase in spine and synapse number is evident within a couple of hours after preparing the slices. Once the initial spine induction has occurred, no further change occurs for up to 13 hr in vitro, the longest time investigated. Thus, the spine increase is occurring during a period when there is little or no synaptic activity during the first hour, and the subsequent stabilization in spine synapse numbers is occurring after synaptic activity returns in the slice. These findings suggest that spines form in response to the loss of synaptic activity when slices are removed from the rest of the brain and during the subsequent 1 hr recovery period. (+info)
Stimulation by 2-deoxy-D-glucose tetraacetates of hormonal secretion from the perfused rat pancreas.
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The effects of alpha- and beta-2-deoxy-D-glucose tetraacetate (1.7 and 8.5 mM) on insulin, somatostatin, and glucagon secretion from isolated rat pancreases perfused in the presence of 8.3 mM D-glucose were compared with those of unesterified 2-deoxy-D-glucose tested at the same two concentrations. The unesterified glucose analog caused, in a concentration-related manner, inhibition of glucose-induced insulin and somatostatin release and augmentation of glucagon secretion. The two anomers of 2-deoxy-D-glucose tetraacetate, however, increased the secretion rate of all three hormones; this effect was also related to the concentration of the esters. No obvious anomeric specificity of the secretory response to 2-deoxy-D-glucose tetraacetate was observed. These findings indicate that the insulinotropic action of hexose esters cannot be accounted for solely by the metabolic effect of their glucidic moieties. They suggest that the A, B, and D cells of the endocrine pancreas are each equipped with a receptor system responsible for the direct recognition of monosaccharide esters as secretagogues. They further support the view that a paracrine effect of insulin on glucagon-producing cells does not represent a major component in the regulation of their secretory activity. (+info)
Endothelin mediates renal vascular memory of a transient rise in perfusion pressure due to NOS inhibition.
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We investigated the renal responses to NO synthase (NOS) inhibition with N-monomethyl-L-arginine (L-NMA; 30 mg/kg) in anesthetized rats in which renal perfusion pressure (RPP) to the left kidney was mechanically adjusted. Acute L-NMA increased blood pressure (BP, approximately 20%) and renal vascular resistance (RVR) rose ( approximately 50%) in the right kidneys that were always exposed to high RPP. In group 1, the left kidney was exposed to a transient increase (5 min) in RPP which was then normalized, and the rise in RVR was similar to the right kidney. In group 2 the left kidney was never exposed to high RPP, and the rise in RVR was attenuated relative to the right kidney. In group 3, rats were pretreated with the endothelin (ET) receptor antagonist Bosentan, immediately before exposure of the left kidney to a transient increase in RPP, and the rise in RVR was also attenuated relative to the right kidney. NOS inhibition resulted in a natriuresis and diuresis in the right kidneys, and approximately 50% of the natriuresis persisted in the left kidney of group 2, in the absence of any rise in RPP. ET antagonism completely prevented the natriuresis and diuresis in response to acute L-NMA in both left and right kidneys. These data suggest that transient exposure to high RPP by NOS inhibition prevents an appropriate vasodilatory response when RPP is lowered, due to the intrarenal action of ET. (+info)
Osmotic shock: modulation of contractile function, pHi, and ischemic damage in perfused guinea pig heart.
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To determine the contribution of changes in extracellular osmolarity to ischemic injury, isolated guinea pig hearts were perfused with hyposmotic (220 mosM) or hyperosmotic (380 mosM) buffer. 31P NMR spectroscopy was used to follow changes in intracellular pH (pHi) and energetics. Hyposmotic buffer decreased myocardial developed pressure by 30 +/- 2% and pHi by 0.02 +/- 0.01 unit, whereas hyperosmotic buffer increased myocardial developed pressure by 34 +/- 1% and pHi by 0.14 +/- 0.01 unit. All hearts recovered to control values on restoration of isosmotic (300 mosM) buffer. The hyperosmolar-induced intracellular alkalosis and developed pressure increase were not prevented by inhibition of Na+/H+ exchange with use of 1 microM HOE-642 but were abolished with use of bicarbonate-free buffers. After 20 min of total global ischemia, hearts perfused with hyposmotic buffer showed significantly greater recoveries of developed pressure, phosphocreatine, and ATP than control hearts, but hearts perfused with hyperosmotic buffer did not recover after ischemia. In conclusion, buffer osmolarities between 220 and 380 mosM alter myocardial pHi and developed pressure but are not deleterious during perfusion. However, buffer osmolarity significantly alters the extent of myocardial ischemic injury. (+info)
Local inhibition of tissue factor reduces the thrombogenicity of disrupted human atherosclerotic plaques: effects of tissue factor pathway inhibitor on plaque thrombogenicity under flow conditions.
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BACKGROUND: Plaque disruption and subsequent thrombus formation lead to acute coronary syndromes and progression of atherosclerotic disease. Tissue factor (TF) appears to mediate plaque thrombogenicity. Tissue factor pathway inhibitor (TFPI) is the major physiological inhibitor of TF. This study analyzes the role of TF on thrombogenicity of disrupted human atherosclerotic plaques and the therapeutic possibilities of its specific inhibition. METHODS AND RESULTS: Human atherosclerotic and normal arterial segments were exposed to heparinized blood at flow conditions modeling medium-grade coronary stenosis in the Badimon perfusion chamber. The antithrombotic effects of the specific inhibition of plaque TF was assessed by reduction in the deposition of radiolabeled platelets and fibrin(ogen) and immunohistochemical analysis of perfused arteries. TF activity was inhibited by both recombinant TFPI and a polyclonal antibody against human TF. Human lipid-rich plaques were more thrombogenic than less advanced atherosclerotic plaques. Specific inhibition of TF activity reduced plaque thrombogenicity, inhibiting both platelet and fibrin(ogen) deposition (580 versus 194 plateletsx10(6)/cm2; P<0.01, and 652 versus 172x10(12) molecules of Fg/cm2; P<0.05, respectively) and thrombosis (immunohistochemistry). CONCLUSIONS: This study documents the key role of TF activity in acute arterial thrombosis after atherosclerotic plaque disruption and provides evidence of the benefit of blocking plaque TF activity. Therefore the inhibition of the TF pathway opens a new therapeutic strategy in the prevention of acute coronary thrombosis after plaque disruption. (+info)
Mild therapeutic hypothermia for postischemic vasoconstriction in the perfused rat liver.
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BACKGROUND: Mild hypothermia, a promising therapy being evaluated for various clinical situations, may suppress the formation of reactive oxygen species during reperfusion and may ameliorate microcirculatory perfusion failure (the "no-reflow phenomenon"). METHODS: Isolated rat livers underwent 30 min of perfusion, 2.5 h of ischemia, and 3 h of reperfusion. The temperature was maintained at 34 degrees C (mild hypothermia, n = 5) or 38 degrees C (normothermia, n = 6) for all three periods by perfusion of a modified Krebs Henseleit solution, air surface cooling, or both. A third group of livers was normothermic before and during ischemia and mildly hypothermic during reperfusion (reperfusion hypothermia, n = 6). Control livers had 3 h of perfusion at normothermia. Chemiluminescence (a measure of the generation of reactive oxygen species) and hepatic vascular resistance were monitored simultaneously to evaluate the effect of temperature on the formation of reactive oxygen species and the development of no reflow. Also measured were thiobarbituric acid reactive species and lactate dehydrogenase, as indicators of oxidative stress and cell injury. RESULTS: Mild hypothermia decreased formation of reactive oxygen species and postischemic increases in vascular resistance. Reperfusion hypothermia also decreased postischemic increases in vascular resistance, but not as effectively as did mild hypothermia. Levels of thiobarbituric acid reactive species were lower for reperfusion hypothermia than for mild hypothermia at only 0 and 30 min of reperfusion. Lactate dehydrogenase was significant only at 0 min of reperfusion for the normothermic group. Oxygen consumption did not change. CONCLUSION: The prevention of hepatic vascular injury by suppression of oxidative stress may be an important protective mechanism of mild hypothermia. (+info)
The vasoconstrictor effect of 8-epi prostaglandin F2alpha in the hypoxic rat heart.
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1. 8-epi prostaglandin (PG) F2alpha, a vasoconstrictor isoprostane, is synthesized under conditions of oxidative stress. This study was undertaken to investigate the vasoconstrictor effect of 8-epi PGF2alpha in the coronary circulation before and after a period of oxidative stress. 2. The effects of the isoprostane 8-epi PGF2alpha and the thromboxane mimetic U46619 were compared in the isolated rat heart perfused in the Langendorff mode at a constant pressure of 80 mmHg. 3. In normal hearts U46619 caused a dose-related reduction in coronary flow (ED50 4.7+/-2.2 nmol). In contrast, 8-epi PGF2alpha had no effect. 4. After reducing perfusion pressure to 20 mmHg for 30 min and reperfusing at 80 mmHg, the dose-response curve to U46619 was unaffected. In contrast, 8-epi PGF2alpha caused a dose-dependent drop in coronary flow (ED50 52.6+/-12.7 nmol), producing a similar maximal reduction to U46619. 5. Similarly, after perfusion with xanthine and xanthine oxidase for either 15 or 30 min there was little change in the response to U46619 in comparison to control hearts. In contrast, 8-epi PGF2alpha caused a reduction in coronary flow similar to that produced by U46619, the magnitude of the response being related to the length of xanthine/xanthine oxidase perfusion. 6. Responses to both U46619 and 8-epi PGF2alpha after xanthine/xanthine oxidase perfusion were blocked by the selective thromboxane receptor antagonist SQ29548 10(-7) M. 7. These results show that oxidative stress in the isolated perfused rat heart reveals a potent vasoconstrictor effect of the isoprostane 8-epi PGF2alpha by an action on the thromboxane receptor. 8. The data also suggest that, since 8-epi PGF2alpha is a partial agonist at the thromboxane receptor, thromboxane receptor reserve is increased by oxidative stress. (+info)
Characterization of airway and vascular responses in murine lungs.
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1. We characterized the responses of murine airways and pulmonary vessels to a variety of endogenous mediators in the isolated perfused and ventilated mouse lung (IPL) and compared them with those in precision-cut lung slices. 2. Airways: The EC50 (microM) for contractions of airways in IPL/slices was methacholine (Mch), 6.1/1.5>serotonin, 0.7/2.0>U46619 (TP-receptor agonist), 0.1/0.06>endothelin-1, 0.1/0.05. In the IPL, maximum increase in airway resistance (RL) was 0.6, 0.4, 0.8 and 11 cmH2O s ml(-1), respectively. Adenosine (< or =1 mM), bombesin (< or =100 microM), histamine (< or =10 mM), LTC4 (< or =1 microM), PAF (0.25 microM) and substance P (< or =100 microM) had only weak effects (<5% of Mch) on RL. 3. Vessels: The EC50 (microM) for vasoconstriction in the IPL was LTC4, 0.06>U46619, 0.05+info)