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Expt. 1 Bioassay of serotonin using rat fundus strip by three point bioassay

This document describes an experiment to determine the unknown concentration of serotonin using a three-point bioassay with an isolated rat fundus strip preparation. The experiment involves constructing dose-response curves for a serotonin standard and test sample, selecting doses that elicit submaximal responses in a 1:2 ratio, and determining the test concentration using the measured responses. Rat fundus tissue is sensitive to serotonin and contracts in a concentration-dependent manner when exposed to increasing doses of the drug. The experiment aims to precisely and reliably estimate the concentration of an unknown serotonin sample through this validated bioassay method.

Education◦
ExperimentNo. 1 Bioassay of serotonin using rat fundus strip by three point bioassay. Mr. Vishal Balakrushna Jadhav Assistant Professor (Pharmacology) GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5
Overview of Discussion • Objective • Principle • Requirements • Experimental specifications (conditions) • Preparation of serotonin stock solution • Preparation of Krebs solution (PSS) • Procedure • Kymographrecording of contractions • Observation table • Calculation • Result and interpretation 2
Objective To determine unknown concentration of serotonin (5- hydroxytryptamine, 5-HT) using rat fundus strip by three point bioassay. 3
Threepoint bioassay 4 In such a type of multiple point bioassay, two responses of standard drug (S1 and S2) and one response due to test sample (T) are taken into consideration. The selection of two responses of standard should be such that they lie on the linear portion of the concentration response curve and also the ratio between the doses should be 1:2. The test response should be intermediate between two responses of standard drug. The precision, reliability and reproducibility of this bioassay are very high. It is most commonly used for estimating the concentration of the unknown sample. Principle
5 Rat fundus is very sensitive tissue for the study of the action of several naturally occuring substances like 5- hydroxytryptamine, histamine, ACh and bradykinin. Unlike the intestinal smooth muscle (ileum), fundus preparation is slow contracting and slow relaxing type of tissue. It is generally employed for the bioassay of serotonin. The fundus (the upper part of the stomach) is grey in colour and therefore, easily identified from the pylorus (pink in colour). A zig-zag preparation of fundus strip is prepared so as to exposed maximum portion of the tissue to drug. The tissue is sensitive to 1 ng/ml of serotonin, 0.05 ng/ml of histamine and 0.2-0.5 ng/ml of ACh.
6 Mechanism of contraction Serotonin acts on 5HT2B- subtype of serotonergic receptor which function through Gq protein and activate membrane bound phospholipase C (PLc) → generating inositol trisphosphate (IP3) and diacylglycerol (DAG), the secondary messengers release Ca2+ intracellularly → cause depolarization and thereby contraction of gastric fundus. Overnight fasted rats are used to record better response of serotonin on isolated rat gastric fundus .
Requirements Animal: Albino rats (150-200 g, overnight fasted) Physiological solution: Krebs solution. Drug- Serotonin (Stock solution: 10 mcg/ml) Chemical- Fixing solution. Instruments: Sherrington recording drum , Student organ bath, Aerator, Insulin or tuberculin syringe to inject drugs in small fractions, Dissecting board and various dissecting instruments. Frontal writing lever and stand, Pipette, Stop watch etc. Miscellaneous: Kymograph paper, plasticin, clips, and thread. 7
Experimentalspecifications(conditions)  Isolated tissue- Isolated rat fundus strip preparation  Drug- Serotonin (Stock solution: 10 mcg/ml)  Physiological salt solution (PSS)- Krebs solution  Time cycle- Total- 5 minutes, Base line- 30 seconds, Contact time- 90 seconds, Washing period- 3 minutes  Applied load/ tension- 1 g  Bath capacity- 40 ml  Bath temperature- 37°C  Speed of rotation of drum- 0.25 mm/ second  Magnification value (Mf) = d (F-W)/ d (F-T)  Aeration- Normal air (1- 2 bubbles/ second) 8
Preparationof serotoninstock solution Stock solution- 10 μg/ml  Dissolve 1 mg of serotonin in 100 ml of distilled water → 10 μg/ml 9
Preparationof Krebs solution (PSS)  Prepare 1 litre of Krebs solution by dissolving NaCl (6.9 g), KCl (0.35 g), MgSO4.7H2O (0.28 g), NaHCO3 (2.1 g), KH2PO4 (0.16 g) and glucose (2.0 g) in distilled water.  MgSO4.7H2O should be added at last.  CaCl2 (0.28 g) should be dissolved separately in distilled water to avoid chances of precipitation of salt.  Mix CaCl2 solution to the higher volume of PSS. 10
Procedure  Sacrifice the rat by a blow on the head and carotid bleeding.  Cut open the abdomen and expose the stomach.  Identify the fundus of the stomach (upper greyish part). Incise it from the junction of pyloric part (pink in colour) and put it in the dish containing Krebs solution.  Incise the fundus from the lesser curvature and open it longitudinally. Give alternate zig-zag cuts to make a fundal strip preparation. Tie both the ends with the thread and mount in the organ bath containing Krebs solution at 37°C. Aerate the tissue.  Apply 1 g load and allow the preparation to equilibrate for 30 min. Using frontal writing lever with 10-12 magnification, record the contractions due to increase concentrations of serotonin. Since the muscle contracts slowly and relaxes slowly, a contact time of 90 seconds and 5 min time cycle are followed to record DRCs of stock and test solutions of serotonin. 11
 Select two doses of standard form the DRC of stock solution of serotonin eliciting sub-maximal responses and bearing a dose ratio 1:2 preferentially and designate them as n1 and n2 elicits the responses S1 and S2 respectively .  Select one dose of test from the DRC of test solution in such a way that the response due to this dose lies preferentially between n1 and n2 and designate it as t elicits the response T.  Record 3 sets of responses due to n1, n2 and t adding them to organ bath in a randomized fashion as per Latin square design mentioned below. The Latin square design of addition of doses is followed to ensure good randomization and to account for the fluctuating sensitivity of the tissue. 12 n1 n2 t n2 t n1 t n1 n2 Latin Square Design -Three point bioassay
 Measure the height of various responses to calculate the mean height of each response (Mean of S1, S2, and T)  Calculate the unknown concentration of serotonin (T) using the following formula and interpret the result. Where, n1 = Lower standard dose n2 = Higher standard dose t = Test dose S1 = Mean of responses of n1 S2 = Mean of responses of n2 T = Mean of responses of t Cs= concentration of serotonin stock (10 μg/ml) 13
Kymographrecordingof contractions 14 Serotonin (10μg/ml) 0.1 0.2 0.4 0.8 1.6 SubmaximalDose Ceiling Dose SupramaximalDose Part I- Dose Response Curve (DRC) of Serotonin (10 μg/ml) Responses Continued 3.2
Kymographrecordingof contractions 15 Serotonin (T) 0.1 0.2 0.4 0.8 1.6 SubmaximalDose Ceiling Dose SupramaximalDose Part II- Dose Response Curve (DRC) of Serotonin (T) Responses Continued 3.2
Kymograph recording of contractions- continue 16 Part III- Recording of responses of n1, n2 & t in three point bioassay. n1- Lower standard dose (0.4ml) n2- Higherstandard dose (0.8 ml) t- Test dose (0.6ml) n1 n2 t n2 t n1 t n1 n2 Latin Square Design Three point bioassay
Observation table- Example 17 Sr. No. Drug (Conc.) Dose (ml) Dose (μg) log (dose)= log (dose in μg ) Response in Conc. of Serotonin in 40 ml organ bath= Dose (μg)/ Bath Capacity (ml) Height (cm/mm) % 1 Serotonin (10 μg/ml ) 0.1 1 0 0.3 16.67 0.025 2 0.2 2 0.3010 0.5 27.78 0.05 3 0.4 4 0.6021 1.4 77.78 0.1 4 0.8 8 0.9031 1.6 88.89 0.2 5 1.6 16 1.2041 1.8 100.00 0.4 6 3.2 32 1.5051 1.8 100.00 0.8 Part I- Dose Response Curve (DRC) of Serotonin (10 μg/ml) 0.00 20.00 40.00 60.00 80.00 100.00 120.00 0 0.5 1 1.5 2 2.5 3 % RESPONSE LOG(DOSE) Log (DRC)of Serotonin Stock (10 μg/ml)
Observation table- Example 18 Sr. No. Drug (Conc.) Dose (ml) Response in Height (cm/mm) 1 n1 0.4 2 n2 0.8 3 t 0.6 4 n2 0.8 5 t 0.6 6 n1 0.4 7 t 0.6 8 n1 0.4 9 n2 0.8 Part II- Dose Response Curve (DRC) of Serotonin (T) (In the way as that of Serotonin stock ) Part III- Recording of responses of n1, n2 & t in three point bioassay.
Calculation 19 Magnificationvalue (Mf) = d (F-W)/ d (F-T) Where- d (F-W) → distance between fulcrumand stylus (writing tip) d (F-T) → distance between fulcrumand point of attachment of tissue Unknown concentration of Serotonin T (μg/ml) Calculate mean of heightsof standard and test responses due to n1, n2 and t Unknown concentrationof Serotonin T (μg/ml)
Result and interpretation  The concentration response curves (CRCs) or dose response curves (DRCs) of serotonin stock (10 μg/ml) and test solution using isolated rat fundus strip preparation was recorded and interpreted graphically as sigmoid or S-shape curves.  The unknown concentration of serotonin (T) using isolated rat fundus strip preparation by three point bioassay was found to be .........μg/ml.  The magnification value was found to be-......... 20
21 Thank You...