In vivo pharmacokinetics using MRS.
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Biomedical subjects
A Sauter
Publications and source records attributed to A Sauter.
Localization of macrophage inflammatory protein: macrophage inflammatory protein-1 expression in rat brain after peripheral administration of lipopolysaccharide and focal cerebral ischemia.
Macrophage inflammatory protein is a member of the C-C subfamily of chemokines, which exhibits, in addition to proinflammatory activities, a potent endogenous pyrogen activity. In this study, we analysed the time-course of expression and cellular source of macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta, in inflammation of the rat brain associated with ischemia and endotoxemia. Using in situ hybridization histochemistry, we observed that intravenously injected bacterial lipopolysaccharide induced a transient expression of macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta messenger RNAs throughout the brain, with maximal expression 8-12 h after lipopolysaccharide treatment. We also revealed an early increase in macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta messenger RNA levels, after permanent and transient middle cerebral artery occlusion, starting as early as 1 h after the occlusion and reaching a peak of expression 8-16 h after middle cerebral artery occlusion. The induction of macrophage inflammatory protein-1 messenger RNA was clearly stronger in the transient than in the permanent middle cerebral artery-occluded rat brains, showing that the reperfusion process influences the extent of the chemokine response after middle cerebral artery occlusion. In situ hybridization combined with immunohistochemistry for glial fibrillary acidic protein, a specific marker for astrocytes, excluded astrocytes as the cellular source of macrophage inflammatory protein-1 messenger RNAs after both middle cerebral artery ischemia and lipopolysaccharide treatment. Using immunohistochemistry, macrophage inflammatory protein-1alpha protein expression was shown to be induced in a time-dependent manner after lipopolysaccharide treatment and middle cerebral artery occlusion. Macrophage inflammatory protein-1alpha immunopositive cells co-localized with cells stained with OX-42 antibody, a microglia/macrophage marker. These results indicate that macrophage inflammatory protein-1 is implicated in the inflammatory reaction of the brain in response to ischemia or infection, and might modulate the host defence febrile response to a pathogenic stimulus.
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Calcineurin inhibitors FK506 and SDZ ASM 981 alleviate the outcome of focal cerebral ischemic/reperfusion injury.
The neuroprotective properties of drugs binding to FKBP12, with and without subsequent inhibition of calcineurin, were investigated in rat models of ischemic embolic stroke. Drug effects on brain infarct volumes evoked by transient middle cerebral artery occlusion (MCAO) and by permanent MCAO were determined in vivo by T2-weighted magnetic resonance imaging and post mortem by triphenyltetrazolium chloride staining and histology. Drugs binding to FKBP12 and inhibiting calcineurin, such as FK506 and SDZ ASM 981, dose dependently reduced the infarct volumes, determined 48 h after MCAO by both magnetic resonance imaging and triphenyltetrazolium chloride staining but only in the transient MCAO model. In vivo potencies to reduce brain infarcts paralleled the in vitro potencies to inhibit calcineurin. Histological staining after 6 days of survival showed that the neuroprotective effects were permanent. Rapamycin, known to bind with similar affinity to FKBP12 but not to inhibit calcineurin, was not neuroprotective but abolished the neuroprotective effects of FK506 when coadministered. In the permanent MCAO models, FK506 showed no effect when injected before and little effect when injected after MCAO. Measurements of core temperatures after MCAO in controls and drug-treated rats do not support hypothermia being the mechanism responsible for neuroprotection. We conclude that drugs inhibiting calcineurin activity are neuroprotective in focal cerebral ischemia/reperfusion but not in permanent ischemia models, possibly by preventing reperfusion injury.
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Cloning of rat HIV-1-chemokine coreceptor CKR5 from microglia and upregulation of its mRNA in ischemic and endotoxinemic rat brain.
Chemokine receptors play a crucial role in the recruitment of immune cells to sites of inflammation. Although chronic diseases of the brain are often accompanied by inflammatory events, there is presently no information about the occurrence and regulation of these receptors in the central nervous system (CNS). Moreover, one CC-chemokine receptor, CKR5, has recently been identified as coreceptor for HIV-1 entry into macrophages. HIV-1 target cells in brain are macrophage-related microglia, which suggests that they are infected by the same mechanism (He et al.,: Nature 385:645-649, 1997). Although rats are not susceptible to HIV-1 infection, they can be used to study chemokine receptor regulation in a variety of brain pathologies. After cloning CC-CKR5 and establishing reverse transcriptase polymerase chain reaction (RT-PCR) for its ligands macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated on activation, normal T cell-expressed and secreted (RANTES), we studied expression of these four mRNAs in purified microglia and compared it with their expression in rat brain. Lipopolysaccharide (LPS)-treated microglia showed transiently increased mRNA levels of both CKR5 and its ligands. Similar data were obtained from brains of LPS-injected rats. In middle cerebral artery occluded (MCAO)-animals, RANTES mRNA was unaffected, whereas CKR5 mRNA showed a sustained rise until 96 hr after surgery. MIPs exogenously added to microglial cultures markedly reduced CKR5 mRNA expression, whereas RANTES did not. MIP mRNAs, in contrast to RANTES and CKR5 mRNAs, were undetectable in normal brain. RANTES appears to play a role distinct from MIPs in brain. In summary, upregulation of CC-chemokines and CKR5 in the CNS upon bacterial infection or in ischemia may impact on microglial activation stage and result in increased risk of HIV-1 infection.
Amino Acid Sequence↗
Susceptibility to cerebral infarction in the stroke-prone spontaneously hypertensive rat is inherited as a dominant trait.
BACKGROUND AND PURPOSE: Susceptibility to cerebral infarction was compared in stroke-prone spontaneously hypertensive (SHRSP), normotensive Wistar-Kyoto (WKY) rats, and F1 hybrids derived from a SHRSP/WKY cross. METHODS: The proximal left middle cerebral artery (MCA) was occluded under anesthesia and infarct volume assessed 24 hours later by magnetic resonance imaging and confirmed 5 days later by quantitative histopathology. Total hemispheric infarct volume was expressed as a percentage of the total brain volume. RESULTS: Infarct volumes measured by MRI in adult SHRSP (19.5 +/- 2.0%) and F1 hybrid rats (19.4 +/- 1.9%) were significantly greater than in WKY (11.1 +/- 2.4; CI [6.07, 10.76]) and (5.93, 10.52), respectively, P<.001). Sensitivity to an ischemic insult was unrelated to blood pressure: although systolic blood pressures differed between young versus adult male SHRSP and between female versus male SHRSP and F1 hybrids, infarct volumes were equal. A close correlation was found between infarct volumes measured by MRI and histology (r=.92, P<.0001). CONCLUSIONS: Outcome to MCA occlusion (MCAO) measured with MRI provides a reproducible and nonterminal quantitative phenotypic marker of stroke susceptibility in the SHRSP. This is the first study to employ MCAO with MRI to quantify stroke susceptibility in F1 hybrid rats and indicates a dominant mode of inheritance for this phenotype.
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K(+)-channels and cytokines as markers for microglial activation.
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Differential and time-dependent expression of monocyte chemoattractant protein-1 mRNA by astrocytes and macrophages in rat brain: effects of ischemia and peripheral lipopolysaccharide administration.
Increasing evidence indicates a key role of chemoattractant cytokines in the accumulation of leukocytes in the central nervous system (CNS) during the course of inflammatory processes. Monocyte chemoattractant protein (MCP-1/JE), a member of the beta-chemokine (C-C chemokine) family, functions as a potent chemoattractant and activator for monocytes. We have investigated the induction of MCP-1 mRNA using in situ hybridization histochemistry (ISH) and characterized its cellular source by combination of ISH and immunocytochemistry in ischemic rat brains as well as in brains of endotoxin-treated rats. Our results show that 6 h-2 d after middle cerebral artery occlusion (MCAO), MCP-1 mRNA is present in astrocytes surrounding the ischemic tissue (penumbra). At later time points (after 4 d), MCP-1 mRNA is found in macrophages and reactive microglia in the infarcted tissue. Peripheral administration of the bacterial lipopolysaccharide (LPS) induced MCP-1 mRNA throughout the brain in a time-dependent manner (1 h-1 d, peak of expression 6-8 h) and was found in astrocytes. In summary, we have found expression of MCP-1 in (a) astrocytes and to a lesser extent in macrophages/reactive microglia after MCA-occlusion and in (b) astrocytes after peripheral administration of LPS. These findings support that MCP-1 is involved in the CNS response to acute trauma or infection and thus may play a key role in inflammatory processes of the brain.
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Analysis of tracer transit in rat brain after carotid artery and femoral vein administrations using linear system theory.
Determination of tissue perfusion rates by MRI bolus tracking methods relies on the central volume principle which states that tissue blood flow is given by the tissue blood volume divided by the mean tracer transit time (MTT). Accurate determination of the MTT requires knowledge of the arterial input function which in MRI experiments is usually not known, especially when using small animals. The problem of unknown arterial input can be circumvented in animal experiments by directly injecting the contrast agent into a feeding artery of the tissue of interest. In the present article the passage of magnetite nanoparticles through the rat cerebral cortex is analyzed after injection into the internal carotid artery. The results are discussed in the framework of linear system theory using a one-compartment model for brain tissue and by using the well characterized gamma-variate function to describe the tissue concentration profile of the contrast agent. The results obtained from the intra-arterial tracer administration experiments are then compared with the commonly used intra-venous injection of the contrast agent in order to estimate the contribution of the peripheral circulation to the MTT values in the latter case. The experiments were analyzed using a two-compartment model and the gamma-variate function. As an application perfusion rates in normal and ischemic cerebral cortex of hypertensive rats were estimated in a model of focal cerebral ischemia. The results indicate that peripheral circulation has a significant influence on the MTT values and thus on the perfusion rates, which cannot be neglected.
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Biphenyl-derivatives of 2-amino-7-phosphono-heptanoic acid, a novel class of potent competitive N-methyl-D-aspartate receptor antagonists--II. Pharmacological characterization in vivo.
A selection of biphenyl-analogues of 2-amino-7-phosphonoheptanoic acid (AP7), N-methyl-D-aspartate (NMDA) receptor antagonists with high affinity in vivo efficacy. The lead compound SDZ EAB 515 was found to inhibit L-phenylalanine uptake by the large neutral amino acid carrier in vitro and in vivo; active transport may thus confer a good bioavailability to this class of compounds. CNS effects were demonstrated by significant changes in 2-deoxyglucose-uptake in various brain regions at doses from 1 to 10 mg/kg i.p. With the most active agent, SDZ 220-581, full protection against maximal electroshock seizures (MES) was obtained at oral doses of 10 mg/kg in rats and in mice. The compound had a fast onset (< or = 1 hr) and a long duration (> or = 24 hr) of action. Motor-debilitating effects (impairment of rotarod performance) occurred at doses about 10 times higher than those required for protection against MES. Neuroprotective activity was demonstrated by the ability of the compounds to reduce the extent of quinolinic acid-induced striatal lesions in rats, in the dose range of 3-15 mg/kg (i.p.) or 10-50 mg/kg (p.o.). In the middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia in rats, the test compounds reduced the infarct size by 40-50% when given i.v. before or by 20-30% when given i.v. 1 hr after MCAO. SDZ 220-581 provided 20-30% protection at > or = 2 x 10 mg/kg p.o. This compound also showed analgesic activity at low oral doses in a model of neuropathic pain, although higher doses were required in model of mechanical inflammatory hyperalgesia. Unexpectedly, SDZ 220-581 at low s.c. doses counteracted the antiparkinsonian effects of L-DOPA in MPTP-treated marmosets. (Sub)chronic administration of SDZ 220-581 did not reduce its ability to protect against quinolinic acid neurotoxicity, and no upregulation of NMDA receptors was detected using a [3H]CGP-39653 binding assay. In conclusion, from a series of biphenyl-AP7-derivatives, SDZ 220-581 is clearly the most active compound in vivo. Its pharmacological profile with a good, long-lasting oral activity might open up novel therapeutic applications for competitive NMDA receptor antagonists.
Amino Acids↗
Expression of tumor necrosis factor alpha after focal cerebral ischaemia in the rat.
Induction of tumor necrosis factor alpha was studied in the brain of rats after focal cerebral ischaemia by occlusion of the left middle cerebral artery. Using a specific antisense riboprobe for in situ hybridization histochemistry, cells positive for tumor necrosis factor alpha messenger RNA were detected within 30 min in the brain regions known to be necrotic within one to two days after onset of ischaemia. Their number increased over a time period of 1-8 h and then declined. Only a few tumor necrosis factor alpha messenger RNA positive cells could be detected four days after the onset of ischaemia. Reverse-transcription polymerase chain reaction experiments showed that maximal increase of tumor necrosis factor alpha messenger RNA level in the ischaemic brain hemisphere occurred 3 h after occlusion of the middle cerebral artery. Immunocytochemical experiments using an anti-tumor necrosis factor alpha antibody showed the presence of tumor necrosis factor alpha immunopositive cells as early as 30 min after occlusion of the middle cerebral artery in the same brain regions where tumor necrosis factor alpha messenger RNA positive cells were detected. Tumor necrosis factor alpha positive cells were highly abundant in the infarcted brain 8-24 h, but only few of them were detectable four days after the onset of ischaemia. Specificity of the anti-tumor necrosis factor alpha antibody and of the induction of tumor necrosis factor alpha protein was confirmed by western blot analysis. Tumor necrosis factor alpha messenger RNA- and protein-positive cells were also detected in the watershed zone and in some structures of the contralateral brain hemisphere. According to their morphology, tumor necrosis factor alpha-positive cells could be identified as microglial cells and macrophages at different states of activation. This assumption was further confirmed by double-labeling studies using the isolectin B4 from Griffonia simplicifolia, a specific microglial/macrophage cell marker. These results demonstrate that expression of tumor necrosis factor alpha is part of an intrinsic inflammatory reaction of the brain after ischaemia.
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Failure of isradipine in cerebral ischemia models.
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A novel EF-hand Ca(2+)-binding protein from abdominal muscle of crustaceans with similarity to calcyphosine from dog thyroidea.
The amino acid sequence of a novel EF-hand Ca(2+)-binding protein from the abdominal muscle of the crayfish, Orconectes limosus, has been elucidated by tandem mass spectrometry and automated Edman degradation. The name CCBP-23 (23-kDa crustacean Ca(2+)-binding protein) is proposed. The protein can also exist as a disulfide-linked homodimer. The sequence of the monomeric form spans 200 residues with an acetylated N-terminal Ser and reveals four EF-hand domains. The 174-mass-unit difference between the calculated average molecular mass of 22,669.6 Da deduced from the sequence and the obtained electrospray ionization mass spectroscopy (ESI-MS) mass of 22,844 Da has not yet been explained. Partial sequence analysis (137 residues) of CCBP-23 from the lobster, Homarus americanus, showed a sequence identity of 74% with the crayfish protein. Homology searches revealed a 44% sequence identity of CCBP-23 from crayfish to calcyphosine, a Ca(2+)-binding protein from dog thyroidea (Lefort et al., 1989). Although CCBP-23 also shows a 44% identity to R2D5 antigen (Nemoto et al., 1993), we believe that both proteins represent two distinct subgroups within the family of EF-hand proteins.
Amino Acid Sequence↗
Strain-dependent drug effects in rat middle cerebral artery occlusion model of stroke.
In the rat middle cerebral artery occlusion (MCAO) model of embolic stroke, calcium antagonists, as well as competitive and noncompetitive N-methyl-D-aspartate (NMDA) antagonists have been studied extensively by various groups. However, there are divergent conclusions concerning their efficiencies. These discrepancies may be due to the different experimental conditions and/or rat strains used. In this study we have assessed in five commonly used rat strains, under identical experimental conditions, the ability of one representative of each of these drug classes to reduce the infarct size in this model. Drugs or corresponding vehicle were injected immediately after permanent unilateral MCAO and the infarct volume was determined 48 hr later by magnetic resonance imaging. In vehicle-treated controls, the total infarct volume was strain-dependent (range, 70-200 microliters), primarily due to variations in the cortex. The extent of total infarct volume reduction (up to 65%) elicited by the three drugs also was strain-dependent, the main effect and variation being observed in the cortex (range, 0-83% reduction). No difference in efficiency was seen between the competitive and noncompetitive NMDA antagonist, whereas the calcium antagonist showed better results in all five strains. The potential to reduce the infarct size in the rat MCAO model with calcium or NMDA antagonists depends upon the strain used. The exact reasons and mechanisms are presently not understood. Such knowledge would certainly add to the confidence in this model and would allow more accurate predictions concerning the clinical efficacy of drugs in stroke trials.
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Cloning of rat interleukin-3 receptor beta-subunit from cultured microglia and its mRNA expression in vivo.
The high-affinity receptors for interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of a ligand binding (alpha-) and a transducing (beta-) subunit. Two distinct transducing subunits (clones AIC2A and AIC2B) have been cloned from mouse, whereas in humans, only one (common) beta-subunit (beta c) has been found. A PCR-based cloning strategy was used to obtain a full-length cDNA sequence from rat microglia including 5'-untranslated regions. Sequence analysis revealed a number of features indicative of the presence of only one beta-subunit in the rat. Most likely, the new rIL-3R beta cDNA is the rat equivalent of human respective murine (AIC2B) beta c subunits. Regulation of rIL-3R beta mRNA expression was investigated in cultured microglia and in vivo. Purified microglia expressed significant amounts of rIL-3R beta mRNA. Addition of lipopolysaccharide (LPS) resulted in a marked upregulation of rIL-3R beta mRNA within approximately 4 hr. No downregulation was observed within 1 week's treatment. No rIL-3R beta mRNA was detectable in normal rat brain. However, 3 hr after a single injection of LPS into the tail vein of a rat, a marked induction of receptor mRNA occurred in a variety of brain regions. Transcriptional rates subsided significantly after 24 hr. rIL-3R beta mRNA was visualized by in situ hybridizations with cRNA antisense probes in ramified cells formerly characterized as microglial cells. rIL-3R beta mRNA was also induced in rat brain after occlusion of middle cerebral artery (MCAO).(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Induction of interleukin-1 beta mRNA after focal cerebral ischaemia in the rat.
The expression of interleukin-1 beta (IL-1 beta) mRNA in the brain in response to cerebral ischaemia in rats was examined using in situ hybridization histochemistry. Focal cerebral ischaemia was induced in spontaneously hypertensive rats by permanent occlusion of the left middle cerebral artery (MCAO). Whereas no IL-1 beta mRNA could be detected in non-operated and sham-operated rats, middle cerebral artery occlusion induced the expression of IL-1 beta mRNA within 15 min in the ischaemic brain regions prone to become necrotic after 1-2 days. The message appeared as spot-like signals, reached a peak after 3 h and then declined to undetectable levels within 4 days. Additionally, a pronounced but brief induction of IL-1 beta mRNA could be detected 1 h after MCAO in the meninges near the watershed zone. The results demonstrate that the inflammatory cytokine IL-1 beta is induced in a time-dependent way after brain ischaemia.
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Determination of creatine kinase kinetic parameters in rat brain by NMR magnetization transfer. Correlation with brain function.
The pseudo first-order rate constant kf of the creatine kinase (CK) forward reaction as well as the CK forward flux FCK,f have been shown to correlate better with cardiac performance than the steady-state levels of ATP and PCr (Bittl, J. A., and Ingwall, J. S. (1985) J. Biol. Chem. 260, 3512-3517). In order to elucidate the relationship between the CK kinetic parameters and brain activity, we have determined, using the non-invasive NMR technique of magnetization transfer, kf and FCK,f in rats, in which brain activity was experimentally varied by administration of either thiopental sodium or bicuculline to decrease or increase electro-encephalogram (EEG) intensity, respectively. The steady-state levels of ATP and PCr, as well as the accumulation of deoxyglucose 6-phosphate (DG-6P) in brain following intraperitoneal administration of deoxyglucose, were determined simultaneously by the NMR technique, whereas the cortical EEG was recorded in a separate experiment. The EEG intensity (range, 1-20 Hz), taken as a measure for brain performance, as well as the amount of DG-6P formed in brain, reflecting the synthesis rate of high energy phosphates (ATP and PCr), linearly correlated with kf. Despite large changes in both EEG intensity (50-250%) and kf (0.12-0.69 s-1) between thiopental sodium- and bicuculline-treated rats, the ATP levels remained constant, whereas the PCr levels decreased with high EEG activity. In contrast to the expectation based on model calculations of CK kinetics, the PCr levels did not increase above control values at reduced EEG intensity (50% of controls). At EEG intensities exceeding control values (bicuculline-treated rats) FCK,f increased as predicted by CK equilibrium. In conclusion, we have shown that in the rat brain, like in the heart, the CK forward rate constant kf, in contrast to ATP and PCr levels, is a sensitive reliable indicator of both increased and reduced function.
Adenosine Triphosphate↗
SDZ ENS 163 a novel pilocarpine like drug: pharmacological in vitro and in vivo profile.
The thiolactone analogue of pilocarpine, SDZ ENS 163, acts in vitro and in vivo as a partial agonist at M1/M3 and as an antagonist at M2 muscarinic receptors. In vitro, the properties of SDZ ENS 163 have been investigated in several functional models for muscarinic receptors: it is a full agonist at M1 (rat superior cervical ganglion, carbachol = 100%) and a partial agonist at M3 receptors (guinea pig ileum). However, the drug shows antagonistic properties at M2 receptors (rat atria). Radioligand binding studies with 3H-N-methylscopolamine (3H-NMS) using CHO cells expressing m1 or m3 receptors indicate that SDZ ENS 163 does not discriminate between m1 and m3 receptors (Ki 1.5 and 2.4 microM respectively). Regarding phosphoinositide (PI) turnover in A9L cells, SDZ ENS 163 is a partial agonist at m1 receptors. In ex vivo neurochemical studies in rats SDZ ENS 163 displays effects characteristic of muscarinic antagonists regarding the turnover of ACh which is increased in the brain. At a similar dose-range SDZ ENS 163 accelerates PI metabolism in the rat brain in vivo and increases the energy of the low frequency band (2-5 Hz) in the rat hippocampal EEG. These effects observed in vivo are consistent with postsynaptic M1 agonistic and presynaptic M2 antagonistic activities. Since SDZ ENS 163 at centrally active doses exerts no peripheral cholinergic effects, it may be useful for the symptomatic treatment of Alzheimer's disease.
Acetylcholine↗
In vivo NMR in pharmaceutical research.
In vivo NMR techniques are currently well established in pharmaceutical research and will likely become increasingly important in the future, as they procure noninvasively morphological, physiological, and biochemical information. The status of magnetic resonance imaging (MRI) and spectroscopy (MRS) in drug development is discussed on the basis of the characterization and evaluation of a rat model of ischemic stroke and the development and profiling of drugs for cerebral ischemia in this model. It can be concluded that MRI is well suited for drug screening (quantitative determination of lesion size), while dynamic MRI and MRS techniques provide relevant information on the mechanism of drug actions. The possibility to follow changes, pathological and therapeutic, in the same individual is important from two points of view. First, variations due to interindividual differences may be eliminated, increasing the statistical power of the results. Second, dose and/or time dependence of a drug can be explored in the same individual. As a result, the number of animals required for a study will be reduced, which from both ethical and economic aspects is highly desirable.
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