Aim

TO study in-vitro pharmacology and physiological salt solutions.

Theory

Introduction to in-vitro pharmacology

Pharmacology is the study of the biological effects of drugs. In vitro pharmacology studies the biological effects of a drug in an isolated environment, such as cell lines or tissues. This setup conveniently eliminates whole organism physiological influences allowing for a detailed analysis of a compound’s impact. In vivo pharmacology is the study of the biological effects of a drug in a complex living organism, and is used to observe the complex physiological effects of a drug.

In vitro (Latin: in glass; often not italicized in English) studies are conducted using components of an organism that have been isolated from their usual biological surroundings, such as microorganisms, cells, or biological molecules. For example, microorganisms or cells can be studied in artificial culture media, and proteins can be examined in solutions. Colloquially called “test-tube experiments”, these studies in biology, medicine, and their subdisciplines are traditionally done in test tubes, flasks, and Petri dishes. In contrast, studies conducted in living beings (microorganisms, animals, humans, or whole plants) are called in vivo.

Results obtained from in vitro experiments cannot usually be transposed, as is, to predict the reaction of an entire organism in vivo. Building a consistent and reliable extrapolation procedure from in vitro results to in vivo is therefore extremely important.

Solutions include: 

• Increasing the complexity of in vitro systems to reproduce tissues and interactions between them (as in “human on chip” systems).
• Using mathematical modeling to numerically simulate the behavior of the complex system, where the in vitro data provide model parameter values.

These two approaches are not incompatible; better in vitro systems provide better data for mathematical models. However, increasingly sophisticated in vitro experiments collect increasingly numerous, complex, and challenging data to integrate. Mathematical models, such as systems biology models, are much needed here.

In isolated tissue preparation, the following precautions should be taken while mounting the tissue

 Tissues with lumen:- e.g. Intestine preparations, seminal vesicles, and vas deferenses, which are not bisected. In the isolated tissue preparation, the two sides of the tissue walls should be sewed in such a way that the lumen of the tissue is not blocked, so that the physiological solution and the drugs will have free access to the lumen. 

Tension on tissue:-  As all tissues in the body are under some tone, it is necessary to exert some tension on the isolated tissue preparations. This depends on the type of muscle used, e.g. in general smooth muscles (fast-moving tissues) like intestine preparations 0.5- 1 gm, 1-3 gm in slow-moving tissues (frog rectus: 1 gm, tracheal chain preparation: 2 gm).

Contracture:- The slow and maintained contraction, which is observed due to the action of an agonist, is called contracture.

Temperature:-  As a frog is a cold-blooded animal, the studies on rectus abdominus are performed at room temperature. A specialized frog Ringer solution is also employed equivalent to 0.6% saline, which is iso-osmotic for frog tissue. All drugs should therefore be used in 0.6% saline.

Physiological solutions:-  These are often referred to as Ringer’s after their discoverer Sydney Ringer. As a general rule, Tyrode’s may be used for experiments with non-innervated muscle whilst Kreb’s is used for nerve-muscle preparation. All physiological salt solutions require aeration, either with an air pump, carbogen (95% O2 and 5% CO2), or with O2 depending on the salt solution used. The buffering capacity of Kreb’s e.g. is completed by the 5% CO2 used in aeration bringing the solution to pH 7.4.

Isolated Uterus:-  The relative proportions of a and B adrenoceptors mediating contraction and relaxation respectively varies from species to species and between estrous and diestrus. In the rat, B- receptor responses remain constant but α receptors appear to be under the action of estrogen. The B- receptor response predominates, but it is sometimes preceded by a transient receptor-mediated contraction when mixed a and ẞ agonists are used. Since the isolated uterus has no inherent tone, relaxation can only be observed by physiological antagonism of the contractile responses to drugs such as ACh. AD is about 100 times more potent in producing relaxation of the uterus than NA. The rat uterus is a good example of a tissue containing ẞ -adrenoceptors. ẞ receptor-mediated relaxation of this preparation can be blocked with ẞ receptor antagonists. 

In vitro pharmacology: physiological salt solutions 

(amounts to be added to 10 liters of distilled water)

*It is increasingly common to use half of this amount of calcium, i.e. 1.25mM instead of 2.5mM.

**CSF = cerebrospinal fluid

A Physiological salt solution/ balanced salt solution (BSS) is a solution made to a physiological pH and isotonic salt concentration. Solutions most commonly include sodium, potassium, calcium, magnesium, and chloride. Balanced salt solutions are used for washing tissues and cells and are usually combined with other agents to treat the tissues and cells. They provide the cells with water and inorganic ions while maintaining a physiological pH and osmotic pressure.

Sometimes glucose is added as an energy source and phenol red is used as a pH indicator. As animal experiments have to be done with isolated organs, it is necessary to use a certain number of physiological solutions of different ionic concentrations which almost act as a substitute for the fluid. They provide isotonicity, and nutrition and act as a buffer when added.

PSS can be defined as the artificially prepared solution to keep isolated tissue alive under experimental conditions. The content of these solutions carries according to tissues & animals taken. These solutions provide food material i.e. energy, O2, and electrolytes in the same proportion as that present in the tissue fluid. They exert the same osmotic pressure as that of interstitial fluid i.e. isotonic with body fluids. Any variant from the principle will lead to shrinkage or blotting depending on hypertonicity & loss of physiological function.

1) . Solution should be prepared carefully with pure material. They should be prepared freshly or can be kept for about 24 hours as they are good media for the growth of microorganisms they must be refrigerated.

The following points should be carefully noted at the time of preparation of the solution:

i). Balance of cations:- Absolute quantity of each ion and preparation among each other especially with Ca+2 & K+ must be maintained.

The common cation and their significance are:

• (a) Na+ ions: Responsible for maintenance of excitability, and contractility rhythmicity of muscles and nerves.
• (b) K+ ions: responsible for increased relaxation of the heart increased neuromuscular transmission and end excitability of nerves.
• (c) Ca++ ions: increase the force of contraction & tone of the heart and decrease the excitability of nervous tissue.

2). Mg+2 ions:- responsible for the contraction of smooth muscles.

i) pH of solution/reaction of solution: pH of various PSS varying from 7.3-7.8 depending upon the organ. At lower pH value tone of preparations tend to decrease & effect of the drug is also altered. pH affects tissue directly and by ionization. At higher pH ionization is less and leads to alkalinity & thus improving cardiac & smooth muscle activity. During the experiment, there can be an accumulation of metabolite which may change the pH. Buffering agents like HCO3- & PO4 are added in saline solution and solutions are changed frequently.

ii) Glucose: Introduced by “Locke” and serves as an energy source, increases the contractility of tissue. It is not an essential constituent for amphibian tissue but indispensable for mammalian tissues.

iii) Distilled Water: It acts as a vehicle to dissolve various ingredients.

iv) Control of temperature: To get a consistent effect, it is important to maintain the temperature of PSS, particularly for mammalian tissue. For instance, when the temperature of the solution is below 370˚C tone of the intestine is decreased, increased the contracts become smaller, and contracts and relaxation time increases.

v) Aeration: Air, O₂, or O2 + 5% CO2 are needed for the proper functioning of the tissues.

Different physiological salt solutions and their uses:

• i) Ringer lock’s solution: It is used in isolated rabbit heart perfusion.
• ii) Frog’s Ringer’s solution: Used in frog’s rectus abdominal muscle and leech dorsalis muscle preparations. 
• iii) Tyrode’s solution: It is used in the experiment of rabbit intestine & guinea pig ileum.
• Iv) De-Jalon’s solution: Used in rat uterus, duodenum colon experiment.
• v) Kreb’s Henseleit solution: Used in guinea pig tracheal chain prep& rabbit’s aortic strip preparation.

Procedure:

• 1. Weigh all the chemicals accurately.
• 2. Dissolve alkalies and sodium hydrogen phosphate, glucose, and calcium chloride separately in Distilled water in a beaker.
• 3. Add the required amount of sodium bicarbonate by dissolving it in a sufficient volume of distilled water.
• 4. Add Sodium bicarbonate at the time of setting up of the experiment (since calcium carbonate is liable to be precipitated if the calcium and bicarbonate are kept long together). 

Precautions

• Add Sodium bicarbonate at the time of setting up of the experiment.
• weigh all the ingredients accurately.
• Due to PPT formation, separate calcium chloride in Distilled water in a beaker.
• The solution should be prepared freshly for every experiment.

VIVA-VOCE

Q.1. What is contractility?

Answer- contractility can be defined as the tension developed and velocity of shortening (ie., the “strength” of contraction) of myocardial fibers at a given preload and afterload. It represents a unique and intrinsic ability of a cardiac muscle to generate a force that is independent of any load or stretch applied.

Q.2. Why sodium bicarbonate is added at the time of setting up of the experiment? Give reason. 

Answer-Sodium bicarbonate produces carbon dioxide when reacts with water, and carbon dioxide produces carbonic acid when reacts with water

Q.3. Why there is a need of providing an isotonic solution?

Answer- An isotonic solution refers to two solutions having the same osmotic pressure across a semipermeable membrane. This state allows for the free movement of water across the membrane without changing the concentration of solutes on either side

Q.4. Define in Vivo and in Vitro techniques.

Answer- In vivo is Latin for “within the living.” In an in vivo experiment, scientists conduct their studies in whole living organisms. The easiest way to remember the difference is that in vivo means inside the living organism and in vitro means outside the living organism.

Q.5. What are the functions of each ingredient in frog ringer solutions?

Answer- Ringer’s solution is a solution of several salts dissolved in water to create an isotonic solution relative to the body fluids of an animal. Ringer’s solution typically contains sodium chloride, potassium chloride, calcium chloride, and sodium bicarbonate. These are used to balance the pH.

Q.6. Which precaution should be taken in isolated tissue preparations? 

Answer- You should avoid excessive pH changes during experiments. 

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