Pharmacological Methods

Spreading depression is a reversible response of brain tissue to a local insult. It has been postulated to be the physiological substrate for the aura phase of classic migraine. The properties and mechanisms of spreading depression were studied in the parietal neocortex of the alfentanil-anesthesized rat, by using a cup electrode that provided close control of the electrical stimulus while allowing specific ion (K+) and potential recordings to be made directly beneath the cathode, the region of origin of the stimulus-induced spreading depression. Cathodal stimulation caused the extracellular K+ concentration to rise, and spreading depressions were observed when this concentration exceeded 8-12 mM in the upper 100-200 microns of cortex. In some experiments extracellular [K+] continued to increase for 5-10 sec after termination of the stimulus, without detectable after-discharge in the potential record, before subsiding. While spreading depression could easily be induced by pressure o...

The effect of several anaesthetic regimens on the increased cutaneous vascular permeability response to histamine and 5-hydroxytryptamine (5HT) was investigated in rats, using the Evans blue dye leakage technique. Diethyl ether administered during injections (2-3 minutes) or urethane 1000 mg/kg did not significantly affect responses to either histamine or 5HT compared with responses obtained in rats given no anaesthetic. However ether administered for 10 or 20 minutes significantly increased the response to histamine. All other anaesthetic regimens tested (urehtane 1250 mg/kg, urethane-chloralose, urethane-pentobarbitone, pentobarbitone, pentobarbitone-xylazine, pentobarbitone-chloralose) reduced responses to histamine and/or 5HT. It was concluded that urethane (1000 mg/kg), although it does not produce full surgical anaesthesia, is the most satisfactory alternative to the use of unanaesthetized animals for dye leakage studies in rats.

A method for stable measurement of ECG in an excised guinea pig heart was presented. The effects of quinidine and its major metabolite, 3-OH quinidine, on QT interval were compared using excised guinea pig hearts with A-V nodal rhythm paced at a constant frequency (190 beats/min). The relative potency of 3-OH quinidine to its parent drug was about one-fourth. Taking into account the fact that the free fraction of 3-OH quinidine in blood is larger than quinidine, this metabolite may contribute to the effects of quinidine in man. Changing the frequency of electrical pacing in excised hearts with A-V nodal rhythm, the RR-QT relation was determined to be rather linear, and this relation became more curvilinear after addition of quinidine (2 mg/liter), suggesting that the Bazett's formula for correcting QT interval with the heart rate is not applicable to guinea pig heart and/or after an addition of drug.

The construction of a system for continuous, automatic monitoring of the activity of many animals is described. The system uses a microprocessor to control sampling of each animal box, accumulate activity scores, keep time, and control printout. For the detection units, infrared light-emitting diodes and infrared-sensitive phototransistors were used. With limited technical advice the system can be constructed by a novice of electronics. A system to monitor 40 animals would take approximately 3 wk to build and would cost about $700.

A package of FORTRAN programs for data entry into NONLIN (version II) is described which may be used in the analysis of plasma-concentration time curves after single or multiple doses for fourteen pharmacokinetic models described in terms of the model's microconstants (volume of compartments, rate constants). In all cases, infusion rates must be zero-order and absorption rates first-order. The program contains an edit subroutine allowing for changes in initial parameter estimates and their lower and upper limits, weighting factors, number of iterations in the NONLIN analysis, and plot option. Individual X and Y values may be changed and listed and X-Y data pairs may be inserted or deleted. Factors pertaining to each of the data entries in relation to NONLIN are discussed.

A polyexponential curve-stripping program, KIN, is described for use on the HP-41CV programmable calculator. The program may be used in the analysis of plasma-concentration-time curves for a three-compartment intravenous bolus or infusion model with linear elimination processes. The coefficients and hybrid rate constants of the exponential function are then used to compute pharmacokinetic parameters (volume of the central compartment, intercompartmental rate transfer constants), which may be used as initial estimates of model parameters in non-linear regression curve-fitting procedures.

Rat liver tryptophan pyrrolase plays a versatile and unique role among hepatic hemoproteins in relation to heme utilization. The depletion of pyrrolase heme in the experimental porphyria produced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine is potentiated by joint administration of any one of 19 drugs known to exacerbate the human disease, but not by any of 13 nonexacerbators. These findings form the basis of a screening test for drug exacerbation of hepatic porphyrias; the conditions, details, and requirements of which are described.

Mast cells degranulation has been assessed by flow cytometry (FACS) taking advantage of the changes in the light scattering properties of mast cells stimulated by secretagogues. In turn, these changes are based on the modification of size, shape, and granule content of the cells before and after stimulation. With FACS, it is possible to work with almost pure mast cell populations (greater than 99%). Moreover, responses to compound 48/80 are carried out in real time and on the same cell sample that acts as internal control. This technique is very sensitive as shown by the ED50 of compound 48/80 (0.051 micrograms/mL) compared to its ED50 on histamine release (0.131 micrograms/mL). The well-known inhibitory effect of disodium cromoglycate against compound 48/80 was clearly observed using FACS. Furthermore, FACS allowed to distinguish between specific degranulating effects and cytotoxicity. Among the secretagogues used, only the degranulation induced by phospholipase A2 was inhibited by...

Using reversed-phase high performance liquid chromatography with electrochemical detection it is possible to measure concomitantly the concentration of several monoamines, their metabolites and aminoacid precursors in 100 microliters of rat cerebrospinal fluid. To study the quantitative relationship between CSF and brain, alterations in brain monoamines and monoamine metabolites were effected by treatment with L-DOPA or L-5HTP administered with or without concomitant inhibition of extracerebral aromatic amino acid decarboxylase and by treatment with alpha-monofluoromethyldopa, probenecid, haloperidol, or probenecid plus haloperidol. The concentrations of the monoamine metabolites, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid, and homovanillic acid as well as of the L-DOPA metabolite, 3-methoxy-4-hydroxyphenylalanine in the cerebrospinal fluid were linearly correlated with the concentrations of these metabolites in the brain. However, these correlations need to be inter...

A method for evaluating passive avoidance- and escape-learning responses simultaneously has been developed for the study of learning and memory in mice. Prolongation of the step-down latency and shortening of the escape latency in the retention test depended on the strength of the voltage of the electric shocks delivered during the training test. Therefore, the step-down latency and escape latency may be good parameters of learning and memory performance. By cycloheximide treatment immediately after training, the step-down latency and escape latency were shortened and prolonged, respectively, in a dose-related manner, and the relationship between the step-down latency and escape latency was significant. Treatment with cycloheximide within 30 min after training caused significant amnesia, but not after more than 60 min. Furthermore, amnesic action of cycloheximide developed 24 hr after the treatment, but not within 4 hr. On the other hand, the step-down latency and/or the escape late...

The pharmacokinetic parameters of muscarinic cholinergic drugs after intravenous (IV) and oral administration to mice was determined with ex vivo (3H)-oxotremorine-M (3H-Oxt) binding to the brain. Oxotremorine had a long duration of action, and arecoline had a short one. There was a significant correlation between the ex vivo ED50 and the in vitro inhibition constants (Ki). Tremorine, a prodrug, inhibited ex vivo binding, but was relatively inactive in in vitro binding. The quaternary amines, methylscopolamine and oxotremorine-M, and the hydrophilic compound, pirenzepine, were relatively weak in inhibiting ex vivo binding because of their poor penetration of the blood-brain barrier. Oxotremorine and BM-5 were similarly bioavailable to the brain by the IV and the oral route. These results indicate that the pharmacokinetic profile of muscarinic cholinergic drugs can be determined with ex vivo (3H)-Oxt binding.

An experimental model of the long QT syndrome has been developed in conscious dogs. This report discusses the methods used in its preparation and the strengths and weaknesses of the model. This new model is suitable for screening the bradycardia-dependent proarrhythmic effects of drugs and for studying the electrophysiology of "torsades de pointes." Permanent bradycardia (RR: 1558 +/- 83 ms) was obtained in 37 dogs by chemically-induced complete atrioventricular block. A 10% further increase of ventricular repolarization (QT: 306 +/- 7.0 ms to 331 +/- 5.5 ms) was obtained in 28 of these dogs by diuretic-induced hypokalemia. Diuretics, despite saline replacement, induced some degree of functional renal failure and extracellular volume losses. The QT interval increased although ventricular cycle length decreased slightly. These biological and electrophysiological parameters were reproducible except for a slow increase in plasma creatinine. Cardiac failure and sudden death ra...

The construction of a system for continuous, automatic monitoring of the activity of many animals is described. The system uses a microprocessor to control sampling of each animal box, accumulate activity scores, keep time, and control printout. For the detection units, infrared light-emitting diodes and infrared-sensitive phototransistors were used. With limited technical advice the system can be constructed by a novice of electronics. A system to monitor 40 animals would take approximately 3 wk to build and would cost about $700.

A method of determining the leakage of 125I-labeled human serum albumin in the plasma into a standardized area of rat skin to study the effects of intracutaneous application of vasoactive substances on microvascular permeability, was reevaluated. The effect is expressed as a quotient (Q) between the amount of labeled albumin in the test area and that in an area injected with buffer. This calculation is simple and as reliable as more complicated expressions of activity. Within a limited dose range, linear/log dose-response curves can be obtained after application of histamine or bradykinin. Locally injected 125I-labeled human serum albumin is eliminated very slowly from rat skin and determination of the amount of radiolabeled albumin in skin after an intravenous injection therefore represents leakage from the vascular compartments. The potentialities and advantages of this method in pharmacological studies are stressed.

Microdialysis in combination with radioimmunoassay was used to study in vivo release of neuropeptides in the rat brain. Microdialysis is a novel brain perfusion technique, by which molecules in the extracellular space may be monitored. This study describes the application to neuropeptide research. A radioimmunoassay procedure was adapted so that substance P- and neurokinin A-like immunoreactivity could be measured in samples collected during in vivo release experiments. The detection limits of substance P and neurokinin A were below 0.1 fmol/100-microliters sample and the IC50 approximately 1 fmol/sample for both peptides. Potassium, introduced via the microdialysis probe, was found to induce a concentration-dependent increase of the extracellular concentration of neurokinin A-like immunoreactivity.

Chronic cigarette smoke exposure in vivo causes decreased conversion of [14C]arachidonic acid (AA) to prostacyclin (PGI2) by isolated aortic tissue and increased conversion to thromboxane (TXA2) by isolated platelets from rats. Alterations in the PGL2/TXA2 balance may be part of the mechanism by which smoking increases the risk of cardiovascular disease. In order to ascertain whether the particulate phase of whole smoke alone could cause these changes, rats were administered smoke condensate in propylene glycol for 56 days via two Alzet (2ML4) osmotic pumps. Pumps containing vehicle, low dose (150 micrograms/hr) or high dose (300 micrograms/hr) condensate were implanted s.c. dorsal to the thoracic vertebrae in male Sprague-Dawley rats. Three-quarters of the condensate-treated rats developed fibrin cysts encapsulating the pumps. Cysts were not seen in vehicle-treated rats. Residual pump contents were weighed and analyzed by GLC to ensure condensate delivery. No significant difference...

A method for studying the acute phase of myocardial infarction in conscious rats has been developed. In preliminary surgery, a loose ligature of atraumatic silk was understitched around the left coronary artery. Its ends were pulled through a polyethylene tube placed within the thorax and fixed under the skin. Seven days later, coronary occlusion was performed by tightening the ligature in conscious animal. Lidocaine and pindolol pretreatment increased the survival rate and attenuated the life-threatening arrhythmias during the first 20 min, but did not influence the infarct size 16 hrs later. An ex vivo perfusion technique for determining the ischemic area has also been developed. 3, 6, and 20 min after coronary ligation, the hearts were excised and perfused with 4% formaldehyde. The ischemic area could not be perfused and remained dark red with a sharp border-line. At the 3rd and 20th min its size was as same as that of the 16-hr infarcted area; however at the 6th min it increased...

A new model for the detection of antidepressant drugs has been described and compared with two other tests. This new model depends on the ability of the antidepressant drugs to selectively modify some behavioral and biochemical changes which occur following bilateral ...

A rapid and reliable method has been developed for the quantitation of nitric oxide by chemiluminescence analysis of headspace gas. Aqueous nitric oxide standards are used to calibrate the method. There is a linear relationship between the amount of nitric oxide and the redox chemiluminescence detector response over the range of 52-2445 pmol of nitric oxide in 2.0 mL of deionized water contained in a sealed 6.2-mL flask. The intra-day and inter-day coefficient of variation values of the method do not exceed 4% and 9%, respectively. The lower limit of quantitative sensitivity and the lower limit of qualitative detection are 52 pmol and 26 pmol of nitric oxide, respectively. This method has been used to measure nitric oxide formation during the incubation of glyceryl trinitrate or sodium nitroprusside in the presence of cysteine. This method has two major advantages over the currently available procedures for the quantitation of nitric oxide, viz., no artifactual formation of nitric o...

Intracerebral microdialysis is a brain perfusion technique in which a tubular, semipermeable membrane perfused with a physiological solution is implanted into a selected brain region. Molecules in the extracellular space diffuse into the perfusate and may be recovered and their concentration determined. Hence, the level of substances such as neurotransmitters may be monitored, and the response to different treatments may be studied. The technique also allows for administration of substances locally to the region of the brain surrounding the perfused tubular membrane. Basic principles of the microdialysis technique are described, and the results from methodological experiments are examined. It is concluded that there is a direct linear relation between the concentration of a molecule in the medium surrounding the dialysis membrane and the concentration measured in the collected perfusate. Relative changes of molecular concentration in brain extracellular space may be calculated even ...

The ability of isolated cardiac muscle preparations to maintain force of contraction was assessed at different extracellular Ca2+ concentrations and at various stimulation frequencies. Preparations were incubated in Krebs-Henseleit buffer at 32 degrees C and electrically stimulated at frequencies between 0.25 and 3.0 Hz. Extra-cellular Ca2+ was maintained at given concentrations between 0.8 and 2.4 mM, a range that included the plasma-ionized Ca2+ levels determined for rat and guinea pig. Developed tension of atrial or papillary muscle preparations of guinea pig heart was more stable at higher stimulation frequencies. Rat atria and ventricular strips were more stable at 0.25 than at 0.5 or 1.0 Hz, and the stability increased at 2.0 and 3.0 Hz. Guinea pig atrial muscle was maintained for a longer time at higher media Ca2+ concentrations; however, no such effect was observed with rat atrial tissue. Thus, the decrementing at developed tension in isolated cardiac muscle preparations is ...

The ligand affinity (Kd) and the number (Bmax) of beta-adrenoceptors in human mononuclear leukocytes was measured using a semiautomatic cell harvester method for the separation of bound and free ligand. The method was first evaluated in terms of contamination, nonspecific filter binding, and well-to-well reproducibility. A comparison was then made with the conventional manual filtration method. The Kd values for the binding of [125lodo]-cyanopindolol to mononuclear leukocyte membranes were independent of the method, whereas the Bmax values were systematically higher (around 30%) when determined with the cell harvester filtration method. A highly significant correlation was found between the Bmax values obtained with both methods.