Isolated goat ileum preparation: An alternative to isolated ileum preparation from laboratory animals

Isolated tissue preparations are commonly used to study the effects of drugs on specific type of receptors. These preparations are also used for bioassay of drugs, characterisation of specific receptor or its subtypes, to determine concentration response curve of an agonist, to study antagonism of drug and in new drug discovery.[1] Isolated heart and rectus abdominis of frog are the two commonly used preparations to teach undergraduate (UG) and postgraduate (PG) students. The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), has instructed universities and institutions not to use frog for teaching purposes, as there is no licensed breeder of frog in India. This has driven us to conduct alternative isolated tissue experiments. Ileum consists of a number of receptors including muscarinic, histaminic, GABAergic, serotonergic, and adrenoreceptors.[2] Guinea pig ileum has been a reference intestinal in vitro preparation to study these changes mediated by receptor. Various receptors including histaminic, GABAergic, and adrenoreceptors are characterised in this tissue preparations. Guinea pig ileum preparations respond to most of the common spasmogens and are particularly suitable for the study of histaminergic receptors.[2] However, it requires some skill and is expensive for routine practical purposes. Isolated ileum preparations from other species like mice and rats are being characterised for interactions between specific receptors and drugs.[3],[4] These preparations are widely used in researches to identify new receptors or to determine the variation in species produced by anticholinergic drugs. To use these preparations for teaching purposes, the laboratory animal species has to be sacrificed. However, goat ileum is a tissue that is easily available from the slaughter house, and animals need not be killed just for teaching purposes. No attempt had been made to study the interactions between drug and receptor in isolated goat ileum preparations, to the best of our knowledge. The present investigation sought to examine the suitability of the easily available and economical goat ileum as a teaching aid for pharmacology teachers, as an alternative to isolated ileal preparation from laboratory animals. This ileum, if found suitable, was meant for UG and PG Practicals. Fresh ileum of healthy, black, male goats were obtained from a slaughterhouse in Tyrode solution. The tissue was transported under ice to the laboratory. The intestinal contents were removed by washing with Tyrode solution, and the mesenteric residues were dissected out. About 2 to 3 cm of ileum was mounted similar to guinea pig ileal preparation described by Kulkarni, 2003[1] with few modifications[2] in an organ bath containing 25 ml of Tyrode solution. Tissues were maintained at 30±1oC, aerated with air and resting load of 1 g during an equilibration of 45 min. Tension adjustments were made as necessary. The tissues were washed every 15 min. Contractions were recorded using frontal writing lever on kymograph. The kymogram was fixed with fixing solution containing colophony in alcohol. Concentration response curve (CRC) to ACh (Sigma chemical Co, St. Louis, MO; USA) was constructed with a contact time of 30 sec. This was followed by three times washing with tyrode solution at an interval of 1 min. The concentrations of ACh (0.2 nM to 0.6 mM, in a fixed volume) were added in geometric progression. The CRC was constructed till ceiling effect to ACh was obtained. In a separate set of experiments, after completing the CRC of Ach, two to three contractile responses of unknown samples of ACh were recorded. The responses in the linear portion of the CRC of ACh were interpolated on the standard graph to obtain the concentration of the unknown. In addition, after completing the CRC of ACh, a separate set of tissues was washed for 30 min with Tyrode solution. It was allowed to equilibrate for 20 min in the presence of atropine (Sigma chemical Co, St. Louis, MO; USA). The antagonistic effect of atropine was determined by constructing the CRC of ACh in the presence of atropine. Responses to ACh were recorded as changes in height from baseline and expressed as percent of maximum response of the ACh. Values are expressed as mean±SEM. The EC50 values for ACh were calculated in the absence or in the presence of atropine using Graph Pad PRISM®, version 4.0. The apparent pA2 value was calculated using the formula pA2 = log [(EC50 of agonist in the presence of antagonist/EC50 of agonist in the absence of antagonist)-1] – log [molar concentration of antagonist]. ACh (0.2 nM to 60 μM) produced a concentration-dependent increase in contraction of isolated goat ileum. [Figure 1] The EC value of ACh was 0.6 nM (n=9). These results suggest 50 that ACh can induce contractions in isolated goat ileum. In the interpolation bioassay method, four different concentrations of unknown namely T1, T2, T3 and T4 were used. Table 1 depicts the concentrations obtained after performing interpolation bioassay, which nearly match with the actual concentrations. Based on these results we recommend that concentration range of 1 to 30 μg/ml be given as test samples to the students. Furthermore, atropine (7 nM) shifted the CRC of Ach towards right with a change in pEC50 value of Ach from 7.207 to 6.205. Atropine (7 nM) caused no reduction in the maximum response of Ach. Therefore, the antagonism produced by