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 chronic conscious, large mammal model for repeated transhepatic studies over a period of 6–8 weeks has been developed for application to the study of the hepatic effects of pancreatic hormone secretion and glucose metabolism, studies of the hepatic mechanisms associated with high first-pass drug metabolism and food interactions, and studies of insulin balance in dogs that have undergone previous pancreatectomy and islet cell auto-transplantation. The preparation of specialized blood sampling catheters and blood flow probes, surgical preparations, pre- and postoperative care, catheter maintenance, and possible complications are described. Data from an oral glucose meal (OMT) are presented. Frequent blood samples from portal, hepatic and jugular veins, and carotid artery were collected and analyzed for plasma insulin concentrations (IRI) and glucose. IRI fluxes were determined for each time period by interpolation of flux X time curves. Total IRI flux was determined as the sum of the areas under the curves for each sampling period. Hepatic insulin extraction was calculated for each sampling interval. By this method of analysis, it was possible to determine hepatic IRI extraction during non-steady-state conditions. The techniques described in the development of this complex animal model, which allows for repeated transhepatic studies in the same conscious subject, may also have application in other chronic studies involving organs other than the liver.
Several methods have been described for the identification and quantification of kininogens based on both immunochemical and functional characteristics. This article presents a rapid, cheap and simple microplate assay of kininogens based on their ability to inhibit cysteine proteases. The target enzyme papain is activated by cysteine HCl and the activated enzyme will be inhibited by added kininogens. The residual enzyme activity that is not inhibited in this reaction subsequently hydrolyzes the added substrate, S-2302, generating a yellow color that is read in a microplate reader at 405 nm. This method is very sensitive, the smallest amount of kininogen that causes significant inhibition of papain is established to be 0.01 micrograms. As a quantitative method, the assay performs accurately when approximately 0.1 micrograms of low molecular weight kininogen or high molecular weight kininogen is added to the test system. The within-run coefficient of variation (%) of the method was 1.7% when the inhibition of papain was in the range 45-70% and the day to day variation as low as 2.3% when performed with a papain inhibition of 80%. Applications of the method are presented, studying chromatographic separated kininogens in plasma, ascites, and urine.
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.
