Methacholine induces wheal-and-flare reactions in human skin but does not release histamine in vivo as assessed by the skin microdialysis technique

A number of investigations have indicated that cholinergic agonists release histamine from isolated mast cells and suggested that cholinergic stimulation releases histamine in vivo. The purpose of this study was to investigate whether the cutaneous wheal-and-flare reaction induced by methacholine challenge in human skin involves histamine release as measured by the skin microdialysis technique. Five hollow dialysis fibers were inserted intradermally in forearm skin in eight healthy subjects. Each fiber was perfused with Kreb's-Ringer bicarbonate at a rate of 3 microliters/min. Dialysates were collected in 2-min fractions before skin challenge and for 20 min after intradermal injection of methacholine 10(-3)-10(-1) M, the vehicle, and a positive control, codeine phosphate 0.3 mg/ml. Histamine was assayed spectrofluorometrically. Methacholine caused a statistically significant dose-related wheal-and-flare reaction, the flare reaction to methacholine 10(-1) M being comparable with that seen with codeine 0.3 mg/ml. No significant histamine release was observed with methacholine, cumulative histamine release of 16 +/- 8 nM by methacholine 10(-1) M being similar to vehicle responses of 15 +/- 9 nM. Histamine release by codeine was 2524 +/- 435 nM. In conclusion, methacholine-induced wheal-and-flare reactions in human skin appeared not to involve histamine release from skin mast cells.     

Measurement of histamine release from human lung tissue ex vivo by microdialysis technique

OBJECTIVE AND DESIGN: Currently no method is available for measurement of mediator release from intact human lung. In this study, a microdialysis technique was used to measure histamine release from mast cells in human lung tissue ex vivo. MATERIAL: Microdialysis fibers of 216 microm were inserted into lung tissue and perfused with Krebs Ringer buffer at a rate of 3 microl/min. After a 15 min period of steady-state perfusion, anti-IgE and vehicle were injected into the lung tissue above individual fibers. Samples from each fibre were collected for 20 min at 2 min intervals. Histamine was assayed fluorometrically. RESULTS: Anti-IgE concentrations of 40-40,000 U/ml dose-dependently released histamine, significant histamine release being demonstrated with anti-IgE concentrations of 400 U/ml and greater. The kinetics of histamine release showed peak values 2-8 min after the injection. Great individual responses were observed but data could be reproduced within individual donors. Monocyte chemoattractant protein-1, a potent basophil secretagogue, did not induce histamine release in lung tissue which indicated mast cells to be the histamine source. Substance P did not release histamine in the lung tissue. CONCLUSIONS: The microdialysis technique allowed measurements of histamine release from mast cells in intact lung ex vivo. The method may prove useful since a number of experiments can be performed in a few hours in intact lung tissue without any dispersion or enzymatic treatment.     

Simultaneous measurement of opiate-induced histamine release in the periaqueductal gray and opiate antinociception: an in vivo microdialysis study

Histamine release and the subsequent activation of H2 receptors in the periaqueductal gray (PAG) are thought to be important components of morphine antinociception. In vivo microdialysis and antinociceptive testing were simultaneously applied in rats to characterize the effects of morphine on PAG histamine release and determine the relationship between histamine release and antinociception. In the absence of nociceptive (tail pinch) testing, morphine (12.8 mg/kg) induced a delayed, long-lasting release of histamine in the PAG. This effect of morphine was abolished by the opiate antagonist naltrexone (1 mg/kg) but was not mimicked by the mu-preferring agonist fentanyl (0.3 mg/kg), suggesting that activation of an opiate receptor other than, or in addition to, the mu receptor is necessary. In contrast to the findings with fentanyl in untested animals, fentanyl combined with nociceptive testing increased histamine release, even though testing alone had no such effect. Unexpectedly, tail pinch testing inhibited morphine-induced histamine release. These results show that the test procedure alters the action of opiates on histamine release, an effect likely to be the result of the stress of repeated tail pinch testing. Therefore, although histamine release may not be obligatory for all types of opiate antinociception, histamine in the PAG may function as a mediator of stress-induced potentiation of opiate antinociception. Even though the microdialysis technique has been acclaimed for its ability to assess neurochemical and behavioral characteristics simultaneously, the introduction of nociceptive testing clearly can alter the neurochemical systems under study.     

Transient increase of blood histamine level induced by pentagastrin. Continuous monitoring by in vivo microdialysis

BACKGROUND: Since few studies of (penta)gastrin-induced histamine release from the gastric mucosa into blood has been performed, an effect of pentagastrin on histamine level of rat blood was examined by using the in vivo microdialysis method. METHODS: Pentagastrin was perfused through the microdialysis probe implanted into the jugular vein of urethane-anesthetized rats or in urethane-anesthetized, totally gastrectomized rats, and dialysis samples of blood were concurrently collected. Histidine decarboxylase (HDC) activities and histamine contents in the glandular stomach and gastric acid output after pentagastrin stimulation were also investigated. RESULTS: Pentagastrin induced a transient increase of blood histamine in a dose-dependent manner but failed to cause any increase of blood histamine in the totally gastrectomized rat. Pentagastrin also induced increases of the HDC activity in the glandular stomach and of the gastric acid output. The peak histamine level in blood occurred 40 min after pentagastrin perfusion, whereas the peak acid secretion occurred after 80-120 min and then leveled off. CONCLUSIONS: The transient increase of blood histamine induced by pentagastrin is attributable to the histamine released from enterochromaffin-like cells and could be monitored by using the in vivo microdialysis method.     

In vivo microdialysis determination of collagen-induced serotonin release in rat blood

We developed a sensitive microbore HPLC method coupled with an on-line microdialysis system to simultaneously measure endogenous 5-hydroxytryptamine (serotonin; 5-HT) and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the rat blood in vivo. A dialysis tube was placed in the right jugular vein. The validity of the procedure is demonstrated because analysis of the aggregating agents, collagen (1 mg/kg) plus epinephrine (0.3 mg/kg) after intravenous injection, showed that they induced an increase in 5-HT and 5-HIAA levels in the jugular vein of the rat.     

Affinity liposomes in vivo: factors influencing target accumulation

To make universal and efficient liposome-based drug carriers, liposomes should be able to recognize and bind other targets beyond their natural targets, the cells of the reticuloendothial system. To make liposomes targeted, numerous methods to couple active substances, primarily, monoclonal antibodies, to the liposome surface have been developed. Resulting immunoliposomes (or affinity liposomes) demonstrate good targeting to cells and organs both in vitro and in vivo. However, the short circulation time of immunoliposomes prevented them from accumulating in targets with diminished blood flow or low antigen concentration. Long-circulating liposomes were prepared by coupling soluble and flexible polymers, such as polyethylene glycol, to the liposome surface. The mechanism of liposome steric protection with flexible polymers is based on the formation of dense "conformational cloud' by a grafted polymer over the liposome surface, and might be analyzed in terms of a statistical model of polymer solutions. By co-immobilization of specific antibodies and protecting polymers on the liposome surface, liposomes can be prepared combining both targetability and prolonged circulation in vivo. A biological model (experimental myocardial infarction in rabbit) was used to estimate the relative importance of different factors (liposome size and coating with protective polymer and/or specific antibody) for effective accumulation of liposomes in the target. Statistical analysis demonstrated that different types of liposomes have to be used in order to reach maximum absolute delivery of liposomes to the target, or maximum target-to-non-target ratio (relative delivery). Therefore, different liposomes should be used as carriers of diagnostic and therapeutic agents.     

Nerve-induced histamine release is of little importance in psoriatic skin

To evaluate the efficacy of systemic ifosfamide, cisplatin (CDDP) combination as first line treatment followed by intraperitoneal (IP) chemotherapy with carboplatin (CBCDA) and etoposide as consolidation in patients with stage III and IV epithelial ovarian cancer. A total of 40 patients with stage III and IV ovarian cancer were entered into the study. Ifosfamide 1 g/m2 plus mesna 1 g/m2 was given as six hour infusion daily for six days and CDDP 75 mg/m2 was given on day seven. Patients completing six cycles of systemic therapy underwent second look laparotomy followed by four cycles of IP chemotherapy with CBCDA 300 mg/m2 and etoposide 200 mg/m2. Of the 40 patients entering the protocol 27 patients completed six cycles with a complete remission (CR) of 65% and overall response of 67.5%. Twenty-two patients underwent second look laparotomy with pathological CR in ten patients, microscopic disease in seven and macroscopic disease in five. Eleven patients completed four cycles of IP chemotherapy. At 52 months was the overall survival (OS) was 36%. The disease free survival (DFS) at 45 months was 38%. Factors affecting OS were ascites (p < 0.011), stage (p < 0.04), weight change (p < 0.017), residual disease (p < 0.001), number of chemotherapy cycles (p < 0.0001) and IP chemotherapy (p < 0.006). Presently 35% patients are alive in CR, 15% are alive with disease, one patients has been lost to follow up while 47.5% have died. Of these four patients had progressive disease, seven relapsed, four died due to treatment related complications and two died in CR due to other causes. Subset analysis of 22 patients who had second look laparotomy and completed four cycles of IP chemotherapy revealed a distinct survival advantage. IFOS + CDDP is an effective combination as first time treatment in advanced ovarian cancer. IP chemotherapy is effective as consolidation and seems to provide a significant survival advantage. Further studies with larger number of patients need to be done to confirm these results.     

The role of ATP-sensitive potassium channels in striatal dopamine release: an in vivo microdialysis study

Inactivation of the retinoblastoma (RB) gene is known to be implicated in the pathogenesis of several types of human cancers. Since structural alterations of the RB gene have not been well examined in human bladder cancer, we looked for mutations in the entire coding region of this gene using polymerase chain reaction (PCR) and single-strand conformational polymorphism analysis of RNA. We also examined allelic loss of the RB gene using PCR-based restriction fragment length polymorphism analysis. Of 30 samples obtained from patients with bladder cancer, eight (27%) were found to have RB gene mutations. DNA sequencing of the PCR products revealed five cases with single point mutations and three cases with small deletions. These mutations included one (10%) of ten low-grade (grade 1) tumours, four (50%) of eight intermediate-grade (grade 2) tumours and three (25%) of 12 high-grade (grade 3) tumours. Likewise, mutations were found in four (21%) of 19 superficial (pTa and pT1) tumours and four (36%) of 11 invasive (pT2 or greater) tumours. In 15 informative cases, loss of heterozygosity at the RB locus was shown in five cases (33%), three cases with RB mutations and two without them. These results suggest that RB gene mutations are involved in low-grade and superficial bladder cancers as well as in high-grade and invasive cancers.     

Inhibitory effect of ketamine on depolarization-induced norepinephrine release in the rat ventral hippocampus investigated by in vivo microdialysis

The release of norepinephrine (NE) in the ventral hippocampus was studied in rats with microdialysis method. The basal release of NE with perfusion of normal artificial cerebrospinal fluid (ACSF) was 1.58 +/- 0.37 x 20 microliters-1 sample. The NE concentration increased significantly with perfusion of high potassium (60 mM) ACSF indicating that depolarization-induced release was up to 5 times higher than the basic level. Ketamine (20 mg.kg-1 and 80 mg.kg-1 im) significantly inhibited the depolarization-induced increase of NE, but did not affect the basal release. Neither 5 mg.kg-1 im of ketamine nor MK-801 had any effect on the basic or the depolarization-induced release. These results suggest that the inhibitory effect of ketamine on the depolarization-induced NE release was not due to the NMDA channel blocking properties of ketamine.     

Time-dependent effects of repeated amphetamine treatment on norepinephrine in the hypothalamus and hippocampus assessed with in vivo microdialysis

The effects of repeated amphetamine (AMPH) pretreatment on norepinephrine (NE) neurotransmission in the hypothalamus and hippocampus were assessed using in vivo microdialysis. Rats were pretreated with either saline or an escalating-dose AMPH regimen (1-->10 mg/kg) over 10 consecutive days, and then were withdrawn from AMPH for either 1 day or 30 days, at which time the animals underwent two consecutive days of testing. As expected, repeated treatment with AMPH resulted in time-dependent changes in both spontaneous locomotor activity and in the psychomotor response to a subsequent challenge injection of AMPH. In addition, repeated exposure to AMPH resulted in time-dependent and regionally-specific changes in the basal concentrations of NE in dialysate, and in the NE response to an AMPH challenge. For example, AMPH pretreatment produced a persistent (at least one month) increase in the basal concentration of NE in the hippocampus, but not the hypothalamus, although the response to an AMPH challenge was altered in both structures. It is suggested that AMPH treatment produces adaptations in NE systems that far outlast the acute effects of the drug, and that these may contribute to both transient and more persistent behavioral sequelae associated with the discontinuation of chronic AMPH use.     

Dopamine D2-receptors regulate neurotensin release from nucleus accumbens and striatum as measured by in vivo microdialysis

The present study was undertaken to examine the role of dopamine D2-receptors in the regulation of neurotensin release. Through a modification of the methods described by Maidment et al. (Neuroscience, 45 (1991) 81-93), we have developed a highly reproducible method of measuring changes in extracellular NT in the striatum and nucleus accumbens by in vivo microdialysis in awake animals. It was observed that calcium-dependent release of NT was evoked in both structures by infusing a high concentration of potassium. In addition, systemic administration of the D2 agonist quinpirole (5 mg/kg) induced a rapid increase of approximately 200% in extracellular NT levels in the lateral caudate and 30-40% in the nucleus accumbens. Conversely, treatment with the D2 antagonist eticlopride (0.5 mg/kg) reduced extracellular NT in the medial anterior caudate and nucleus accumbens 20-30%, but had no effect in the lateral anterior caudate. These data demonstrate for the first time that D2-receptors are important in the dopaminergic regulation of extrapyramidal and limbic NT release in conscious animals.     

Effects of Ca2+ channel antagonists on striatal dopamine and DOPA release, studied by in vivo microdialysis

The chaetotaxy of argentophilic structures on three species of the monogenean genus Gyrodactylus was investigated in an attempt to distinguish species of this genus. Maps were prepared for Gyrodactylus salaris from Scandinavia and compared with two native species of Gyrodactylus parasitising salmonids in Britain, namely Gyrodactylus derjavini and Gyrodactylus truttae. The maps were subsequently refined and analysed for zones of homology and differentiation. The results demonstrate that G. salaris can be readily distinguished by this technique, which is, therefore, of great potential value in the identification of this notifiable pathogen. The key aggregations of sensilla discriminating G. salaris are, ventrally, the antero-ventral set, the medio-lateral set and the postero-lateral set, and, dorsally, the postero-dorsal set.     

Modulation of pentagastrin-induced histamine release by histamine H3 receptors in the dog

BACKGROUND: The histamine H3 receptor has been shown to inhibit pentagastrin-induced gastric acid secretion in dogs. Since pentagastrin releases histamine in dogs, we have now assessed whether the effects of H3-receptor ligands may be indirectly mediated by changes in gastric histamine release. METHODS: Pentagastrin infusions (1 or 6 micrograms/kg/h), alone or together with the H3-receptor agonist (R) alpha-methylhistamine (1.2 mumol/kg/h) or the antagonist thioperamide (0.1 mumol/kg/h), were performed in dogs. One group (anaesthetized) was used for enzyme immunoassays of plasma histamine and, when required. (R) alpha-methylhistamine in the gastrosplenic vein, and another group (non-anaesthetized) for measurement of gastric acid secretion. RESULTS: Histamine levels were increased five- and eight-fold after 1 and 6 micrograms/kg/h pentagastrin, respectively, whereas acid output was nearly maximal at the lower dosage. (R) alpha-methylhistamine, at a plasma concentration of 0.15 microM, inhibited histamine release by 78% (P < 0.007) and 37% (not significant) and the total acid output by 44% (P < 0.05) and 19% (not significant) after infusion of 1 and 6 micrograms/kg/h pentagastrin, respectively. Thioperamide, together with pentagastrin in low dose, significantly increased histamine release by 212% (P < 0.05), whereas acid output increased by 34% (not significant). CONCLUSIONS: The histamine H3 receptor mediates a negative feedback control of pentagastrin-induced release of gastric histamine. It is tonically activated by endogenous histamine after pentagastrin in low dosage. The control of acid secretion by the H3 receptor seems to involve modulation of endogenous histamine release, possibly by means of enterochromaffin-like cells.     

NMDA receptor-mediated pilocarpine-induced seizures: characterization in freely moving rats by microdialysis

1. Pilocarpine administration has been used as an animal model for temporal lobe epilepsy since it produces several morphological and synaptic features in common with human complex partial seizures. Little is known about changes in extracellular neurotransmitter concentrations during the seizures provoked by pilocarpine, a non-selective muscarinic agonist. 2. Focally evoked pilocarpine-induced seizures in freely moving rats were provoked by intrahippocampal pilocarpine (10 mM for 40 min at a flow rate of 2 microl min(-1)) administration via a microdialysis probe. Concomitant changes in extracellular hippocampal glutamate, gamma-aminobutyric acid (GABA) and dopamine levels were monitored and simultaneous electrocorticography was performed. The animal model was characterized by intrahippocampal perfusion with the muscarinic receptor antagonist atropine (20 mM), the sodium channel blocker tetrodotoxin (1 microM) and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine maleate, 100 microM). The effectiveness of locally (600 microM) or systemically (10 mg kg(-1) day(-1)) applied lamotrigine against the pilocarpine-induced convulsions was evaluated. 3. Pilocarpine initially decreased extracellular hippocampal glutamate and GABA levels. During the subsequent pilocarpine-induced limbic convulsions extracellular glutamate, GABA and dopamine concentrations in hippocampus were significantly increased. Atropine blocked all changes in extracellular transmitter levels during and after co-administration of pilocarpine. All pilocarpine-induced increases were completely prevented by simultaneous tetrodotoxin perfusion. Intrahippocampal administration of MK-801 and lamotrigine resulted in an elevation of hippocampal dopamine levels and protected the rats from the pilocarpine-induced seizures. Pilocarpine-induced convulsions developed in the rats which received lamotrigine perorally. 4. Pilocarpine-induced seizures are initiated via muscarinic receptors and further mediated via NMDA receptors. Sustained increases in extracellular glutamate levels after pilocarpine perfusion are related to the limbic seizures. These are arguments in favour of earlier described NMDA receptor-mediated excitotoxicity. Hippocampal dopamine release may be functionally important in epileptogenesis and may participate in the anticonvulsant effects of MK-801 and lamotrigine. The pilocarpine-stimulated hippocampal GABA, glutamate and dopamine levels reflect neuronal vesicular release.     

The electrical characteristics of human skin in vivo

PURPOSE: The objectives of this study were to investigate the impedance properties of human skin in vivo and to examine the effect of iontophoresis upon them. METHODS: Having established the intra- and inter-individual variation in basal values of skin impedance, the effect of varying iontophoretic current density, ionic strength and counter-ion on the rate of recovery of skin impedance after iontophoresis was investigated. RESULTS: Passage of an iontophoretic current caused a significant reduction in the magnitude of the skin impedance. Increasing the current density caused an even greater reduction in the value of the skin impedance and slowed the rate of recovery. Reduction of the ionic strength resulted in an increase in the rate of recovery following iontophoresis. A significant increase in the rate of recovery was observed when CaCl2 replaced NaCl as the electrolyte. Although visual inspection revealed the presence of greater erythema when CaCl2 was used, there was an absence of the mild sensation experienced by volunteers when using NaCl. The last part of the study established a correlation between transepidermal water loss and impedance analysis as complementary methods for probing skin barrier function in vivo. The data were fitted to an equivalent circuit consisting of a resistor in parallel with a constant-phase element and a mechanistic model proposed to explain the electrical properties of the skin. CONCLUSIONS: The first comprehensive investigation of the effect of iontophoresis on the electrical properties of human skin in vivo has been described. It would appear from the results, and from their interpretation, that impedance spectroscopy may be an effective method to quantify the impact of iontophoresis on the skin, and to determine the extent to which proposed drug delivery regimens will perturb skin barrier function.     

Measurement of carbamazepine and carbamazepine epoxide in the human brain using in vivo microdialysis

We report the first study of carbamazepine and carbamazepine-10,11-epoxide concentrations determined by using intracerebral microdialysis in three patients undergoing depth electrode studies for the evaluation of medically intractable epilepsy. Very small microdialysis catheters, affixed to and inserted with the depth electrodes, sampled drug concentration in the extracellular environment. We perfused artificial extracellular fluid continuously, and varied the perfusion rate to permit estimation of the absolute drug concentration in the extracellular space. Serum samples were obtained simultaneously. The relation between dialysate and extracellular concentration (recovery fraction) depended, in vivo but not in vitro, on the relative lipophilicity of the compounds, suggesting that diffusion of the drug within the brain is a major determinant of microdialysate drug concentration. When this is taken into account, the steady-state extracellular concentrations of these compounds closely mirror their unbound serum concentrations.     

In vivo monitoring of norepinephrine and hydroxyl free radical generation by ferrous iron in the myocardium with a microdialysis technique

1. We examined in vivo monitoring of norepinephrine and hydroxyl radical generation in rat myocardium with a microdialysis technique. For this purpose, we designed the microdialysis probe holding system which includes loose fixation of the tube and synchronization of the movement of the heart and the probe. 2. The hydroxyl free radical (.OH) reacts with salicylate and generates 2,3- and 2,5-dihydroxybenzoic acid (DHBA) which can be measured electrochemically in picomole quantity by high performance liquid chromatography (HPLC). 3. After probe implantation, norepinephrine concentration of dialysate decreased over the first 150 min and then reached an almost steady level. A positive linear correlation between the ferrous iron and .OH formation trapped as 2,3-DHBA (R2 = 0.960) and 2,5-DHBA (R2 = 0.982) was observed using the microdialysis technique. 4. The present results indicate that non-enzymatic oxidation in the extracellular fluid may play a key role in hydroxyl radical generation by ferrous iron.