Mice were housed in a cage containing a T maze. A watering place was located at the entrance of the maze. The right and left arms of the maze each had two exits, one of which led to the home cage where food was placed, while the other led to the watering place via a bypass. The exit leading to the home cage in either the right or left arm was alternately closed every 90 min. One-way swinging doors were inserted at the entrance to each arm and between each bypass and the watering place. The mice were given a cholinergic neurotoxin--ethylcholine mustard aziridinium ion (AF64A)--(8 nmol) or saline as a control into the left ventricle 2 weeks before they were housed in the apparatus. Those mice housed in this apparatus mastered the alternation task at a 5-sec delay on day 3 in the sham group and on day 4 in the AF64A group. When a longer delay (5-90 sec) was introduced for the mice that mastered the alternation task at 5-sec delay, the AF64A group made significantly more errors than did the sham group at 60- and 90-sec delays. These results show that the apparatus is useful in estimating working memory in mice with little effort.
A modified perfusion apparatus for electrophysiological, hemodynamic and metabolic studies on hearts of small animal (e.g., rat, guinea pig) has been described. The apparatus consisted of two oxygenators connected to the aortic cannula of the Langendorff perfused heart, each one of which could be used on demand. The constant aortic perfusion pressure was controlled by oxygenating gas (5% CO2 in O2, usually). The perfusing medium could be changed easily and quickly without interruption. Left ventricular pressure was recorded by means of a balloon-catheter, while special suction electrodes obtained the high-amplitude, noise-free electrogram recordings. The coronary effluent partial pressure of oxygen (Po2) was continuously monitored, which enabled the calculation of myocardial oxygen consumption (MVO2). The effluent partial pressure of carbon dioxide (Pco2) and pH value were also measured simultaneously. A computerized system of data acquisition, calculation, storage, and end report has also been described. In this study, the influence of indomethacin on hypoxia-induced hemodynamic and metabolic changes in the isolated guinea pig heart was observed. The advantages and disadvantages of the described and standard Langendorff apparatus have been discussed also.
A quantitative method to assess relative potencies (IC50) of 5-lipoxygenase (5-LO) enzyme inhibitors was established in antigen-induced contractions of tracheas isolated from actively sensitized guinea pigs (Schultz-Dale model). The relative potencies of four purported 5-LO inhibitors determined in this tissue assay were compared with those from a crude enzyme preparation isolated from guinea pig neutrophils. All compounds suppressed ovalbumin (OA)-induced tracheal contractions in a concentration-related manner in the presence of indomethacin and pyrilamine. IC50 Values, determined from the percent inhibition values obtained from responses at 30 ng/mL OA of these compounds ranged from 0.56–15 μM. A similar rank order of potency for inhibition of 5-HETE formation from a crude enzyme preparation was observed. This suggested that these agents had a common mechanism of action in the two assay systems and further validated the IC50 values determined in trachea assay. LY171883, an LTD4/LTE4 receptor antagonist, also suppressed OA-induced contractions concentration dependently with an IC50 of 4.9 μM determined by this method. LTD4 concentration-response curves were not altered by any of the four 5-LO inhibitors, ruling out the possibility that these agents were acting as LT receptor antagonists. Results of this study demonstrated that relative potencies of 5-LO inhibitors can be quantitatively assessed using this airway tissue model, which helps in identifying potential therapeutic agents for asthma.
Many physiological changes take place during pregnancy, and the disposition profile of endogenous and exogenous compounds may change, too. Thus knowledge of the disposition pattern of a compound may be useful in relation to its therapeutic effect(s) and its potential toxicity on the fetus and the newborn. Because the amount of a compound received by the fetus is a product of placental transfer rate, and available maternal amount, and because it is difficult to control and evaluate the factors that may affect such a transfer in women, we set up an in situ perfused placental model in the rabbit. The reliability of the model was borne out by comparing the placental transfer of theophylline with antipyrine, a commonly used marker of placental exchange, at steady state after a two-step infusion at mean arterial plasma concentrations of 8 and 5 mg/L, respectively for theophylline and antipyrine. The rabbit placenta was perfused in situ with a modified Earle's buffer at a 1-mL/min flow rate. During perfusion, maternal plasma, placental perfusate, biochemical parameters, gas exchange, body temperature, and electrocardiogram were carefully monitored. The maternal plasma and perfusate drug concentrations over time were fitted by appropriate models and kinetic parameters were calculated. Umbilical vein/maternal artery concentration ratios reached equilibrium soon after the loading infusion was stopped for both drugs. Placental clearance averaged 0.62 and 0.77 mL/min for theophylline and antipyrine, respectively, and the clearance index of theophylline was 0.81 +/- 0.07. Although human and rabbit placentas are structurally dissimilar, the rabbit placenta perfused in situ appears to be a useful preparation for measuring the transfer processes and the related and governing factors, of different compounds.
Assessment of cardioactive substances is usually performed using animal tissue, with the effects being extrapolated to humans, thereby potentially introducing errors due to species differences. In order to validate the use of human atrial tissue, known positive and negative inotropic agents were tested on trabeculae obtained from patients' atrial appendages at the time of cardiac surgery requiring, cardiopulmonary bypass. Trabeculae were selected according to strict criteria: cross-sectional area less than 1.0 mm2, resting force (RF) less than 0.7 g, and developed force (DF) greater than 0.8 g. Each trabecula received only one drug in a cumulative dose manner. Where necessary, the vehicle used to dissolve or stabilize the drug solution was also tested. In addition, the relative DF of "no-drug," "time-only" controls were measured during the same time period. After adjusting for the effect of time on the preparation, relative DF was increased to 157% by dobutamine (1.5 x 10(-5) M), to 136% by amrinone (5.6 x 10(-4) M), and to 117% by ouabain (2 x 10(-7) M). The relative DF decreased with nifedipine and propranolol, with 50% inhibition for both drugs being 1.5 x 10(-7) M. Although human ventricular muscles might be more appropriate to use in order to determine the effects observed with the whole heart, they are extremely difficult to obtain on a regular basis. The results of this study show that the atrial trabecular preparation offers an acceptable alternative.
A digital timing system has been developed to control the airflow from a respiration pump used in small animal pulmonary measurements. Three separate periods of up to 10 sec may be selected with millisecond resolution using thumb-wheel switches. A modular design using CMOS technology minimized the number of interconnections among the various parts of the system. A larger number of timing channels, longer time periods, and greater time resolution may be achieved by directly extending the existing design.
We examined the effect of dithiothreitol (DTT) on the isolation and calcium transport properties of rat cardiac sarcoplasmic reticulum (SR). DTT at a concentration of 4 mM was included in the homogenization/initial isolation and the high-salt (KCI) buffers used in the preparation of the SR fraction. The inclusion of DTT in the SR isolation media did not affect the total amount of SR protein isolated from ventricular tissue. However, SR isolated in the presence of DTT exhibited a significantly increased (p < 0.05) rate of calcium transport at free calcium concentrations ranging from 0.1 μm up to 2.0 μm, and a 26% increase in the apparent affinity of the SR calcium transport system for Ca2+. DTT included in the calcium-transport reaction medium did not significantly alter the rate of SR calcium transport in SR preparations isolated in the absence or presence of DTT. These results demonstrate that functionally important sulfhydryl groups of the SR calcium transport pump may be oxidized during the isolation of the membrane fraction and that such oxidation can be reduced by including DTT in the isolation buffers.
Restrained conscious rats have been widely used for physiological and pharmacological hemodynamic studies. In this condition, the variability of the circulation is unclear. Repeated measurements in restrained normal rats showed stable systemic hemodynamics (cardiac output ranging from 130 +/- 14 to 174 +/- 10 mL/min, mean arterial pressure ranging from 109 +/- 9 to 117 +/- 5 mmHg) and splanchnic hemodynamics (splanchnic blood flow ranging from 8.51 +/- 1.89 to 13.01 +/- 1.45 mL min-1 100 g-1 body wt) over a period of 1 hr. Slight but not significant hemodynamic variations, however, occurred in pulmonary blood flow. Similarly, plasma noradrenaline concentrations did not vary over this period (plasma noradrenaline level ranging from 186 +/- 36 to 358 +/- 64 pg/mL). These plasma noradrenaline concentrations were similar to those measured in a group of conscious unrestrained rats 3 hr after recovery from surgery (292 +/- 60 pg/mL). A significant correlation was observed between plasma noradrenaline concentration and heart rate.
A simple and reliable high-performance liquid chromatographic method is described for the simultaneous determination of histamine (His), which cannot be directly oxidized, and noradrenaline (NA), which can be directly oxidized within the useful working potential range. The isoindole products formed by precolumn derivatization of His and NA with o-phthalaldehyde (OPA) and 2-mercaptoethanol (2-ME) yielded a linear relationship of detection between the electrochemical signal and the compound content to a minimum detectable limit of 50 pg (signal-to-noise ratio = 3:1) for both compounds at 0.5 nA of detector range. Without 2-ME, OPA derivatives of both His and NA were not detectable electrochemically at the oxidation potential range from 0 to +1 V. Although the peak potential was +0.85 V for both His and NA, we used +0.7 V for both compounds to keep background noise minimal. The capacity factors of some electrochemically interfering compounds were also determined. The significance of OPA/2-ME derivative of NA is discussed relative to the direct oxidation of catecholamines. An example of a practical application of the method to the determination of His and NA in rat cardiac tissue is presented.
An attempt was made to use neuroblastoma cells for testing neuroprotective drug effects. To achieve cellular damage, cytotoxic hypoxia was induced in neuroblastoma cells after 10 days in culture by addition of sodium cyanide (1 mmol/L) to the culture medium and was terminated after 6 hr by replacing the cyanide-containing fresh nutrient medium. During this hypoxic period cells were additionally deprived of glucose. They were allowed to recover for another 7 days. Drugs were available to the cells from 30 min prior to hypoxia until 24 hr after hypoxia. Cell concentration of high-energy phosphates and culture protein content were determined as representatives for the posthypoxic development of cell damage, cell activity and viability. While barbiturates and phenytoin revealed neurotoxic effects when applied in doses higher than 300 mumol/L, chlorpromazine, dizocilpine, ketamine, ketazocine, naftidrofuryl, and flunarizine protected neuroblastoma cells against hypoxic damage. These results were comparable to those obtained from primary cultures of neurons under similar experimental conditions. In addition, they were in keeping with neuroprotective drug effects obtained from in vivo experiments. It is suggested that neuroblastoma cells are suitable for testing neuroprotective drug effects.
A method for studying the in vivo accumulation of inorganic mercury along the nephron of Sprague-Dawley rats pretreated with a radiolabelled 0.66 μmol/kg dose of mercuric chloride is described in this article. Forty-eight hr after rats received the radiolabelled dose of mercuric chloride intravenously the kidneys of the animals were perfused in situ with a collagenase solution in order to dissect and isolate various readily assessable segments of the nephron and collecting duct. Three different categories of tubular segments were isolated; proximal convoluted tubules, proximal straight tubules and combined segments of the distal nephron and collecting duct. A group of isolated tubular segments were measured in length, drawn up and placed in counting tubes, and placed in a gamma counter for the determination of the content of inorganic mercury that accumulated in them during the 48 hr subsequent to the administration of the dose of mercuric chloride. In a separate set of animals, the intrarenal distribution of inorganic mercury was determined 48 hr after the intravenous dose of mercuric chloride was asministered. Inorganic mercury accumulated mainly in the renal cortex and outer stripe of the outer medulla. In addition, the concentration of inorganic mercury in the outer stripe of the outer medulla was twice that in the cortex. The findings obtained with the isolated tubular segments revealed that most of the accumulated inorganic mercury in the kidneys of the rats was in the proximal tubule. The content of inorganic mercury in the proximal straight tubules was significantly greater than that in the proximal convoluted tubules. Negligible amounts of inorganic mercury were detected in the distal segments of the nephron and collecting duct. The data obtained from the isolated tubular segments indicate that the increased accumulation of inorganic mercury that occurs in the outer stripe of the outer medulla is due specifically to increased accumulation of inorganic mercury in the proximal straight tubules. The method used to study the accumulation of inorganic mercury in segments of the nephrons proves to be sensitive enough to explain the mechanism for various patterns of accumulation of inorganic mercury in the kidneys of rats.
A formula derived by Gero and Tallarida (1977) relates the equilibrium dissociation constant of a partial agonist (P) and that of a second agonist (A) of greater efficacy that acts on the same receptor. The second agonist may or may not be a strong agonist. Accordingly, if the dissociation constant (K) of one of the compounds is known, say from the method of partial irreversible receptor blockade, then the dissociation constant for the other may be determined from the complete concentration-effect curves of the compounds and the derived formula: kp = KA (Ap-Ai)Pi/(Ap + KA)Ai, where Pi and Ai are equieffective concentrations of P and A, Ap = the concentration of A that gives an effect = the maximum effect of P. The practical use of this formula is illustrated here for several agonists, and for each, the value of K obtained is compared to that obtained by partial irreversible receptor blockade. In all cases tested, the agreement is quite good, thus suggesting that this method may be a practical alternative.
The inorganic dye, ruthenium red, is an ammoniated form of ruthenium oxychloride. The purity of commercial samples of ruthenium red has been confused as commercial vendors often provide a “dye content” figure for their product, which some investigators have equated with purity. However, dye content is the same as ruthenium content and is not related to purity, although it will affect the relative molecular weight of this compound. As a result, concentrations of ruthenium red used in various biochemical studies have been calculated based on the supposed impurity of the commercial product, a product purified in the laboratory of the investigator, or by simply ignoring this purity question. Purity of four different commercial products as well as two different “purified” materials was determined by comparison of absorbance spectra and extinction coefficients. Taking into account differences in the relative ruthenium content of each preparation of ruthenium red, the results demonstrate no significant differences between these materials indicating that commercially available samples of ruthenium red are essentially pure.
Assessment of endothelial integrity is an obligatory step in many pharmacological studies. Integrity of endothelium is affected by manipulations performed during the removal and cleaning of the vessel and by some of the silver-staining techniques utilized for demonstrating interendothelial junctions. When aortas were cleaned of periadventitial tissue in cold Tris-saline (once separated from the animal) by untrained personnel, only 45% of the endothelium was preserved. When cleaning was performed in situ by trained personnel while flushing with cold Krebs-Ringer-6% albumin, over 95% was left intact. AgNO3-staining performed before fixation produced a 50% loss of endothelium when using NH4Br and (NH4)2S as developers. AgNO3-staining performed after fixation produced over 95% recuperation of endothelium when 2% glutaraldehyde, 150 mM NaCl, 40 mM phosphate buffer, pH 7.4, were utilized as initial fixative, NH4Br and (NH4)2S being equally effective as developers. Chloride ions were necessary to intensify silver lines. Several patterns of deendothelization were produced by mechanical and chemical injury with saponin, NH4Br and (NH4)2S. In all cases, hematoxylin staining was employed as an auxiliary technique to interpret images of injured endothelium. Presence of albumin protected the endothelium from mechanical damage.
Primary cultures of adult rat hepatocytes were established using two different isolation procedures: a two-step collagenase perfusion and a method using ethylenediaminetetraacetate (EDTA) as the dissociating agent. Both techniques provided good yields of hepatocytes with comparable viability. The evolution of hepato-specific protein levels and several drug-metabolizing enzyme activities were followed for 8 days in cultured hepatocytes obtained by both methods. EDTA-isolated hepatocytes maintained a low gamma glutamyltransferase (GGT) activity, whereas collagenase-treated cells acquired a high GGT level. Transferrin secretion and tyrosine aminotransferase (TAT), alanine aminotransferase (ALT), and microsomal epoxide hydrolase (mEH) activities were stable in both EDTA- and collagenase-isolated hepatocytes, whereas albumin secretion, aspartate amino transferase (AST) activity, total cytochromes P-450 content, IA1 and IIB1 P-450 isoenzymes, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) levels, and bilirubin glucuronidation decreased faster in collagenase-treated cells. The most important difference observed was the maintainance of the mixed-function oxidase system in EDTA-isolated hepatocytes. These results emphasize the critical role of isolation technique in stabilization of differentiated hepatocytes in primary culture.
A bioassay for the measurement of platelet-activating factor (PAF) based on the quantification of platelet aggregation was developed. The method used a platelet analyzer in conjunction with a multiwelled micromixing device and whole blood collected from male New Zealand White rabbits. This bioassay can be nonselective and used to quantitate platelet aggregation induced by any activator. The EC50 for platelet-activating factor, arachidonic acid, and adenosine diphosphate were 0.0232, 55, and 10 microM, and at these concentrations the half maximal aggregation response occurred at 7.5, 10.0, and 12.5 min, respectively. The bioassay was capable of the sensitive quantification of platelet aggregation in small volumes (50 microL) of citrated rabbit whole blood, using a short (i.e., 15-min) incubation period. This enabled multiple bioassays to be performed without the need for a large volume of whole blood to be collected from the rabbit. Selective measurement of platelet-activating factor was achieved by adding inhibitors of arachidonic acid- and adenosine diphosphate-dependent pathways of platelet activation, that is, acetylsalicyclic acid and phosphoenolpyruvate/pyruvate kinase, respectively, to the citrated rabbit whole blood immediately before bioassay. These inhibited arachidonic acid- and adenosine diphosphate-induced platelet aggregation, but had no effect on platelet aggregation induced by platelet-activating factor. Platelet-activating factor was selectively inhibited by its receptor antagonist BN 52021. This method for measuring platelet-activating factor was reproducible; at the EC50, the inter- and intrabioassay coefficients of variation were within acceptable limits at 13.17% and 9.75%, respectively.
Comparison between drug effects in MPTP- and non-MPTP-treated marmosets following intracerebroventricular (ICV) and intraputamen administration are given in the present report. Both side-effect profiles and the ability to reverse MPTP-induced hypokinesia were assessed using a variety of dopamine receptor agonists (quinpirole, PHNO, ADTN), cholinergic antagonists (scopolamine, secoverine, himbacine), a glutamate receptor antagonist (MK801) and a 5-HT receptor agonist (8-OH-DPAT). Our results show that direct infusion of agents into the putamen or via the ICV route can reverse parkinsonian-like symptoms in marmosets. Furthermore, chronic implanatation of cannulae into the putamen or ventricles of nonhuman primates can be useful in assessing the therapeutic activity of agents that do not readily cross the blood-brain barrier.
A fluorimetric and a colorimetric method for the determination of hydrogen peroxide (H2O2) production by isolated cells were compared. Despite a higher sensitivity of the fluorimetric assay, using homovanillic acid (HVA) as reagent, a significantly lower H2O2 production by rat pleural macrophages was measured in comparison to the colorimetric phenol red method. A negative influence of HVA on H2O2 production was detected in the colorimetric assay. These results suggest that the fluorimetric assay with HVA is unsuitable for determining H2O2 formation by isolated cells.
Myocardial infarction studies in pigs have been complicated by the use of antiarrhythmic drugs or the high incidence of ventricular fibrillation. We report on a new model of experimental myocardial infarction in thiamylal-anesthetized Yucatan minipigs. These studies were performed in the absence of intravenous antiarrhythmic drugs. No animals required resuscitation during either surgery or reperfusion and only 19% were resuscitated during occlusion. Extensive systemic hemodynamic, regional contractility and coronary blood flow measurements were continuously obtained during left anterior descending coronary artery (LAD) occlusion (45 min) and reperfusion (240 min). Mean arterial blood pressure and left ventricular + dP/dt decreased during occlusion, and both declined further upon reperfusion. Persistent dysfunction (segmental shortening from 24.8 +/- 1.3 to 3.9 +/- 0.9% (p less than 0.001); pre-occlusion and 5 min post-occlusion, respectively) occurred immediately after occlusion in the myocardium perfused by the LAD, while late declines in segmental shortening (19.6 +/- 0.9 to 17.2 +/- 1.2%; pre-occlusion and 240 min post-reperfusion, respectively) were observed in myocardium perfused by the left circumflex coronary artery. While heart rate did not change during occlusion, tachycardia occurred at the onset of reperfusion. Although initial reactive hyperemia following reperfusion was manually inhibited, high LAD blood flow following reperfusion occurred early (0 to 60 min) but returned below pre-occlusion values late (180 to 240 min). The area at risk represented 23.1 +/- 0.9% (n = 34) of the left ventricle and 39.0 +/- 3.2% of this area was infarcted. Therefore, 9.2 +/- 0.9% of the left ventricle was infarcted. These data suggest that myocardical infarction in anesthetized minipigs can be achieved without the aid of intravenous antiarrhythmic drugs and reduced cardioversion. Therefore, this new model can be utilized in the evaluation of therapeutic compounds focused on altering the detrimental consequences of myocardical infarction.
