Early delineation of ischemic tissue in rat brain cryosections by high-contrast staining.
BACKGROUND AND PURPOSE: After short periods of ischemia, commonly used staining methods yield only moderate differences in optical contrast between normal and damaged brain tissue when gray-scale images are used for computer-assisted image analysis. We describe a high-contrast silver infarct staining (SIS) method that allows an early delineation of ischemic tissue as soon as 2 hours after middle cerebral artery occlusion (MCAO) in rat brain cryosections. METHODS: Rats were subjected to permanent MCAO for 2, 4, 6, and 48 hours. The optical densities were quantified in nonischemic white and gray matter and in damaged tissue from gray-scale images of serial sections with the use of a video camera-based image analyzing system. SIS, hematoxylin-eosin, Nissl, and nitroblue tetrazolium stainings were performed in cryosections, and 2,3, 5-triphenyltetrazolium hydrochloride (TTC) staining was performed in unfrozen vibratome sections. In addition, the range of reduced cerebral blood flow (CBF) in areas demarcated by SIS was determined in iodo[14C]antipyrine autoradiograms of adjacent cryosections. RESULTS: At all times after MCAO, only SIS showed significantly (P<0.01) lower optical densities in damaged than in normal brain tissue for both white and gray matter. TTC staining was as effective as SIS 6 and 48 hours after MCAO. The tightest correlation between areas of reduced SIS and of reduced CBF was found at a mean ischemic CBF of 22.3 mL/100 g per minute. This corresponds to a CBF range of 0 to 44 mL/100 g per minute in areas of reduced SIS. CONCLUSIONS: In contrast to other staining methods, SIS allows a reliable delineation of ischemic brain tissue (core plus penumbra) from nonischemic white and gray matter of rat brain cryosections as soon as 2 hours after MCAO. (+info)
Cerebral blood flow responses to somatosensory stimulation are unaffected by scopolamine in unanesthetized rat.
Studies with positron-emission tomography have indicated that muscarinic acetylcholine receptors may be involved in the mechanism of enhancement of cerebral blood flow (CBF) by neuronal functional activation. We examined the effects of muscarinic receptor blockade by scopolamine on the local CBF responses to vibrissal stimulation in the whisker-to-barrel cortex sensory pathway in unanesthetized rats. Local CBF was measured by the quantitative autoradiographic [(14)C]iodoantipyrine method. Scopolamine (0.4 or 0.8 mg/kg) was injected i.v. 30 min before measurement of local CBF; control rats received equivalent volumes of physiological saline. Vibrissae on the left side of the face were stroked continuously throughout the 1-min period of measurement of CBF. Local CBF was determined bilaterally in four structures of the pathway, i.e., spinal and principal sensory trigeminal nuclei, ventral posteromedial thalamic nucleus, and barrel field of the sensory cortex, as well as in four representative structures unrelated to the pathway. The higher dose of scopolamine raised baseline CBF in the two trigeminal nuclei, but neither dose diminished the percentage of increases in local CBF because of vibrissal stimulation in any of the stations of the pathway. These results do not support involvement of muscarinic receptors in the mechanism of enhancement of local CBF by functional neuronal activation, at least not in the whisker-barrel cortex sensory pathway in the unanesthetized rat. (+info)
Cerebrovascular reactivity to CO(2) and hypotension after mild cortical impact injury.
Cerebrovascular reactivity to CO(2) or hypotension was studied in vivo and in vitro [pressurized arteries ( approximately 82 micrometer) and arterioles ( approximately 30 micrometer)] at 1 h after mild controlled cortical impact (CCI) injury in rats. The cortical perfusion response [assessed using laser-Doppler flowmetry (LDF)] to altered CO(2) was diminished (up to 81%) after mild CCI injury. The responses to CO(2) alterations in arteries and arterioles isolated from the injured cortex were similar to responses in vessels isolated from sham-injured animals. After mild CCI injury, the autoregulatory response to hypotension (measured using LDF) was maintained or even enhanced, depending on the method used to measure the response. Vessels isolated from the injury site showed a response to changes in pressure similar to that in vessels isolated from sham-injured rats. We conclude that mild CCI injury produces complicated alterations in cerebrovascular control. Whereas the autoregulatory response to hypotension was maintained or even enhanced, the in vivo vascular response to CO(2) was severely compromised. The altered response to CO(2) was not caused by an intrinsic vascular perturbation but rather an altered milieu after mild CCI injury. (+info)
Direct detection of antipyrine metabolites in rat urine by (13)C labeling and NMR spectroscopy.
Antipyrine is a useful probe to evaluate variation of in vivo activities of oxidative hepatic drug-metabolizing enzymes. Here we describe a new approach using (13)C labeling and NMR spectroscopy for the direct and simultaneous detection of all phase I and phase II metabolites of antipyrine in rat urine. [C-methyl-(13)C]Antipyrine was synthesized and administered orally to rats (100 mg/kg), and the 0- to 24-h postdose urine was analyzed by 100-MHz (13)C NMR spectroscopy under the conditions of distortionless enhancement by polarization transfer without any pretreatments such as deconjugation, chromatographic separation, and solvent extraction. Consequently, all the major metabolites in urine were successfully detected with favorable signal-to-noise ratios in the limited acquisition time (30 min). The assignments of the resonances were performed by enzymic modification and spiking authentic samples. The reproducibility of the NMR detection was sufficient for the quantitative evaluation of the metabolic profile. Effects of 3-methylcholanthrene on antipyrine metabolism were examined by this approach to evaluate variation of in vivo phase I and phase II metabolism of antipyrine in rats. The present approach is useful and practical to evaluate variation of in vivo activities of conjugation enzymes as well as oxidation enzymes responsible for the formation of antipyrine metabolites in rats. This direct approach would enhance the value of the antipyrine test because of the simplicity and convenience. (+info)
Decreased antipyrine clearance following endotoxin administration: in vivo evidence of the role of nitric oxide.
Klebsiella pneumoniae endotoxin has been found to decrease hepatic P450-mediated drug-metabolizing enzyme activity in a time-dependent manner. In this study, we investigated the role of nitric oxide (NO) in the decrease in hepatic drug-metabolizing enzyme activity caused by endotoxin in vivo. We measured in vivo pharmacokinetic parameters of antipyrine in rats treated with endotoxin and/or a selective inhibitor of inducible NO synthase (iNOS), S-methylisothiourea. Intraperitoneal injection of endotoxin (1 mg/kg of body weight) dramatically decreased the systemic clearance of antipyrine, reflecting reduced hepatic drug-metabolizing enzyme activity, and significantly increased the level of nitrite and nitrate (NOx) in the plasma. S-Methylisothiourea (10 mg/kg) reversed this decreasing antipyrine clearance and reduced the level of NOx in plasma. Repeated injections of an NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK-409; 10 mg/kg), at a dose which maintained plasma NOx at the same levels as those caused by endotoxin injection, also decreased the systemic clearance of antipyrine. These findings suggest that the overproduction of NO observed in this animal model is at least partially responsible for the significant reduction in the hepatic drug-metabolizing enzyme activity that may happen in a gram-negative bacterial infection. (+info)
Modifications of blood volume alter the disposition of markers of blood volume, extracellular fluid, and total body water.
Recirculatory pharmacokinetic models for indocyanine green (ICG), inulin, and antipyrine describe intravascular mixing and tissue distribution after i.v. administration. These models characterized physiologic marker disposition in four awake, splenectomized dogs while they were normovolemic, volume loaded (15% of estimated blood volume added as a starch solution), and mildly and moderately hypovolemic (15 and 30% of estimated blood volume removed). ICG-determined blood volumes increased 20% during volume loading and decreased 9 and 22% during mild and moderate hypovolemia. Dye (ICG) dilution cardiac output (CO) increased 31% during volume loading and decreased 27 and 38% during mild and moderate hypovolemia. ICG-defined central and fast peripheral intravascular circuits accommodated blood volume alterations and the fast peripheral circuit accommodated blood flow changes. Inulin-defined extracellular fluid volume contracted 14 and 21% during hypovolemia. Early inulin disposition changes reflected those of ICG. The ICG and inulin elimination clearances were unaffected by altered blood volume. Neither antipyrine-defined total body water volume nor antipyrine elimination clearance changed with altered blood volume. The fraction of CO not involved in drug distribution had a significant effect on the area under the antipyrine concentration-versus-time relationships (AUC) in the first minutes after drug administration. Hypovolemia increased the fraction of CO represented by nondistributive blood flow and increased the antipyrine AUC up to 60% because nondistributive blood flow did not change, despite decreased CO. Volume loading resulted in a smaller (less than 20%) antipyrine AUC decrease despite increased fast tissue distributive flow because nondistributive flow also increased with increased CO. (+info)
Ketamine distribution described by a recirculatory pharmacokinetic model is not stereoselective.
BACKGROUND: Differences in the pharmacokinetics of the enantiomers of ketamine have been reported. The authors sought to determine whether these differences extend to pulmonary uptake and peripheral tissue distribution and to test the hypothesis that tissue distribution of the stereoisomers differs because of carrier-mediated drug transport. METHODS: The dispositions of markers of intravascular space and blood flow (indocyanine green, ICG) and total body water and tissue perfusion (antipyrine) were determined along with S-(+)- and R-(-)-ketamine in five mongrel dogs. The dogs were studied while anesthetized with 2.0% halothane. Marker and drug dispositions were described by recirculatory pharmacokinetic models based on frequent early and less-frequent later arterial blood samples. These models characterize pulmonary uptake and the distribution of cardiac output into parallel peripheral circuits. RESULTS: Plasma elimination clearance of the S-(+)-ketamine enantiomer, 29.9 ml x min(-1) x kg(-1), was higher than that of the R-(-)-enantiomer, 22.2 ml x min(-1) x kg(-1). The apparent pulmonary tissue volumes of the ketamine S-(+) and R-(-)-enantiomers (0.31 l) did not differ and was approximately twice that of antipyrine (0.16 l). The peripheral tissue distribution volumes and clearances and the total volume of distribution (2.1 l/kg) were the same for both stereoisomers when elimination clearances were modeled from the rapidly equilibrating peripheral compartment. CONCLUSIONS: Although the elimination clearance of S-(+)-ketamine is 35% greater than that of the R-(-)-enantiomer, there is no difference in the apparent pulmonary tissue volume or peripheral tissue distribution between the stereoisomers, suggesting that physicochemical properties of ketamine other than stereoisomerism determine its perfusion-limited tissue distribution. (+info)
Evolution of microcirculatory disturbances after permanent middle cerebral artery occlusion in rats.
Nonischemic brain capillaries show a continuous and heterogeneous plasma perfusion. In the current study, plasma perfusion was investigated in rats during 2 to 168 hours of permanent middle cerebral artery occlusion. Perfused capillaries were detected in brain cryosections by fluorescein isothiocyanate (FITC) dextran after 10 minutes of circulation time. Heterogeneity of capillary perfusion was identified by Evans blue (EB), which circulated for 3 seconds. In this setting, the heterogeneity of intracapillary EB concentrations reflects heterogeneities in capillary flow velocities. The CBF was quantified by simultaneous iodo[14C]antipyrine autoradiography. When moving from normal flow to low-flow areas in the ischemic hemisphere, three states of capillary filling could be distinguished: state 1--fast perfusion, filling by FITC dextran and EB (CBF 0.33 mL x g(-1) x min(-1)); state 2--delayed perfusion, only FITC dextran filling (CBF 0.104 mL x g(-1) x min(-1)); state 3--minimal perfusion, no dye filling (CBF 0.056 mL x g(-1) x min(-1)). In tissue of state 1 at the borderline to ischemic tissue, a higher heterogeneity of intracapillary EB concentration (85.7%) was found than in the contralateral nonischemic hemisphere (76.4%) (P < 0.05), indicating a compromised microcirculation. The adjacent ischemic areas were filled by FITC dextran (state 2) 2 to 4 hours after middle cerebral artery occlusion, indicating a maintained, although slow, perfusion at this time. Later, minimal perfused areas (state 3) progressively replaced the delayed perfused areas (state 2). This study shows, for the first time, the evolution of microvascular disturbances in relation to CBF. In the low-flow areas, an early residual plasma perfusion is later followed by a lack of perfusion or minimal perfusion. In areas of higher, although reduced flow at the border between normal and ischemic tissue, an extreme capillary perfusion heterogeneity indicates permanent microcirculatory abnormalities. (+info)