Organ Baths - Virtual Labs PDF

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In this year the labs were virtual - we had weekly tutorials based on the laboratory process. This document is specific work for the organ baths lab and provides enough theory for a student to understand what will be required of them in the practical set up....

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Virtual Lab Notes

Archana Arya-Ram

Organ Baths: Organ baths provide an ideal way to expose isolated tissue to a known drug concentration and measure responses that are produced. Organ baths are designed to replicate the in vivo situation, providing several important conditions to keep the tissue alive while also allowing it to produce measureable responses. What is an organ bath? → An apparatus used to suspend tissue of interest in an aqueous solution that can mimic a physiological fluid → Different tissues are attached to hooks which can then be treated ex vivo with drugs and responses measured → In pharmacology labs we use organ baths that have 20mL capacity which means 20mLs of solution surrounds the tissue at one time → Typical solution = Krebs solution: mix of glucose Na, K, Ca, Mg and Cl that nourishes the tissue, keeping it alive → O2 tube is present to allow constant aeration of the Krebs so the tissue has an O 2 supply → Composition of gas = 95% O2 and 5% CO2 → Temperature must be maintained at 37℃ in order to keep the tissue alive → This is provided by that water-jacket that surrounds the organ bath and has a constant flow of warm water running through the system to warm the Krebs and maintain temperature Measuring responses in organ baths: To allow us to measure a response from the tissue there needs to be a link between the organ bath and the computer. On of the most common responses examined involves the contraction of muscle cell. This could be skeletal muscle (e.g. gasterocnemius), cardiac muscle (i.e. part of the heart) or smooth muscle (e.g. ileum, aorta). Measurement of contractions is via the tissue hooks and the force transducer. Tissue is suspended in the organ bath between two hooks, one of which is connected to a force transducer. Once the tissue is in the organ bath a resting (i.e. the tissue is not contracting) tension is applied. This tension gently stretches the tissue so that any contraction will result in a downward tug on the force transducer, but is not so strong that the tissue cannot contract against it. Why use an organ bath? The recording of the contractile responses from isolate tissues and the study of effects of drugs alone, and of the effects of drugs on the contractile responses to endogenous compounds, agonists and to nerve muscle stimulation is one of the most fundamental and enduring techniques used by pharmacologists. Three types of important information can be obtained from the recording of responses from isolated tissues: 1. The responses of an isolated tissue to endogenous compounds and to nerve r muscle stimulation may be characterised a. Each tissue or piece of tissue is unique in its pattern of innervations and endogenous receptor profile. Consequently each tissue or piece of tissue responds differently to nerve or muscle stimulation and to the addition of endogenous compounds. By studying the effects of drugs, with known mechanisms of action, on the contractile responses of an isolated tissue, the characteristics of the contractile responses of that isolated tissue to nerve muscle stimulation and to endogenous compounds are

Virtual Lab Notes

Archana Arya-Ram

determined. Subsequently, the characteristics of the contractile responses to nerve or muscle simulation and to endogenous compounds f isolated tissues (in vitro effects) are used to predict the characteristics of responses of organ systems intact animals (in vivo effects). b. While contractile responses are often measured using an organ bath, other types of data can also be obtained. Because the organ bath is an enclosed volume of fluid tissue within it can be stimulated, then the fluid (physiological salt solution) can be collected and analyse to investigate what compounds have been released from the tissue (e.g. neurotransmitter, ions). 2. The mechanism of action of a new drug may be studied a. In order to improve the drug treatment of illness, new drugs are continually being developed either by extraction from natural sources or synthetically. Once a purified sample of a new drug has been obtained, its mechanism of action and actions in animals have to be determined. The mechanism of action of a new drug is determined by studying the effect of the new drug alone of isolated tissues, as well as the effect the new drug has on the contractile responses of isolated tissues to nerve or muscle stimulation, endogenous compounds and agonists. The effects of a new drug are compared to the effects of drugs with known mechanism of action. Finally, the mechanism or action of the new drug is deduced. 3. The potency of a new drug may be determined a. One of the determinants of the dose of a drug used to treat human illness is the potency of the drug. The potency of agonists and antagonists may be determined by recording the resultant contractility from selected isolated tissues under appropriate experimental conditions and constructing concentration-response curves. This can also help to clarify the type of agonism or antagonism the drug produces. Advantages and disadvantages: As with all experimental techniques, organ baths have their advantages and disadvantages. Organ baths allow the examination of drug effects on a single organ/tissue, without the need to consider pharmacokinetic (ADME) processes. Tissue can be exposed to a known concentration of drug and a response measured, without worrying about the potential loss of drug through stomach acidity or the first-pass effect or how long the drug will remain at effective concentrations before elimination processes predominate. The use of isolated tissue can also minimise the number of animals that need to be used. For example, a single guinea-pig can provide ileum for up to 30 experiments, each investigating multiple drug concentrations. The downside of this is that an organ bath is less like an actual in vivo situation, so provides minimal insight into clinically relevant doses. The concentration-response curves can provide useful information about mechanism of action (e.g. what receptor(s) does the drug work at) and can provide a broad indication of potential dosage, but if the organ baths are being employed as part of a drug discovery process, testing in whole organisms is required prior to use in humans. The use of isolated tissue is relatively cost-effective after an initial outlay for equipment, with the need for tissue, physiological salt solution and carbogen being the main expenses. With ongoing developments in the field of cell culture, organ baths are being used less commonly in research due to the limited capacity for high-throughput experiments and the availability of cell lines which can effectively replace the need for animal experiments in some studies....