Solve Organ Bath Problem with Guinea-Pig Ileum & Dog Mast Cells

  • Thread starter MiniJo
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In summary: To test for potential receptor blockade on the ileum or histamine degradation, prepare an organ bath, and add the ileum, a known histamine agonist, and the leaves. If the ileum contracts, we know that the leaves are responsible for degradation of histamine (since the agonist was able to be received). If there is no contraction, then the leaves are blocking the receptors. (Note that when I say "leaves" I actually mean some property of the leaves).4. To test inhibition of release, set up another organ bath, and add the dog mast cells and the ileum. First as a control experiment, let the two things sit in the bath, making
  • #1
MiniJo
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Homework Statement


A botanist friend of yours has returned from a trip to the rain forest. She brings with her a package of dried leaves which contains what native healers believe to be an antidote to severe itching. You have access to an organ bath set up in which you can set up an isolated guinea-pig ileum to record responses to added agonists as well as a dog mastocytoma-derived cell line that responds to stimuli with the release of histamine. How would you use these preparations to explore the potential mechanism underlying the putative therapeutic effects of the leaves?

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The Attempt at a Solution


Basically, this question is asking for the possible histamine-related effects of the leaves, if there are any. So I identified different stages to the experiment so far.

1. Control experiment.
The ileum has histamine receptors, therefore it will contract if it binds with histamine. Knowing this, I put the ileum into the organ bath and added histamine. Ideally this will produce a contraction.

2. Addition of leaves.
The leaves are believed to have antihistamine properties. So in another organ bath, I added the ileum, histamine and the leaves. I'm not sure what the best order for these additions is. Anyway. If the ileum stops contracting, then the leaves are inhibiting the reception of histamine or it's degrading the histamine. If the ileum continues to contract, then I'm not sure what's happening... maybe the leaves inhibit synthesis or release of histamine. We'll test this later.

3. To test for potential receptor blockade on the ileum or histamine degradation, prepare an organ bath, and add the ileum, a known histamine agonist, and the leaves. If the ileum contracts, we know that the leaves are responsible for degradation of histamine (since the agonist was able to be received). If there is no contraction, then the leaves are blocking the receptors. (Note that when I say "leaves" I actually mean some property of the leaves).

4. To test inhibition of release, set up another organ bath, and add the dog mast cells and the ileum. First as a control experiment, let the two things sit in the bath, making sure there's no contraction. Then stimulate the mast cells (UV, chemical agent...etc), this would cause degranulation, allowing the ileum to contract. In another organ bath, add the dog mast cells, the ileum and the leaves. Stimulate the mast cells to degranulate. If the ileum does not contract, then the leaves are interfering with degranulation (release of histamine). If the ileum does contract, then the leaves are ineffective against histamine.

5. To test for the inhibition of synthesis of histamine, we can add dog mast cells and ileum to an organ bath, stimulate the cells to degranulate and measure the level/amount of contraction in the ileum. Then, rinse the bath and add the leaves and mast cells immediately after. (These mast cells should have a lot less histamine compared to the beginning since they've just degranulated). Let the bath sit for a period of time. Add the ileum, then stimulate the mast cells again and measure the level of contraction in this second time around. If the level of contraction in the ileum is significantly less in the 2nd trial than in the first, then synthesis of histamine is inhibited by the leaves.

Does this seem like a proper solution?
If you see any flaws or have extra suggestions, please do tell me. I greatly appreciate any help. =)
 
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  • #2
You appear to be well along the right track here.
MiniJo said:
2. Addition of leaves.
The leaves are believed to have antihistamine properties. So in another organ bath, I added the ileum, histamine and the leaves. I'm not sure what the best order for these additions is. Anyway. If the ileum stops contracting, then the leaves are inhibiting the reception of histamine or it's degrading the histamine. If the ileum continues to contract, then I'm not sure what's happening... maybe the leaves inhibit synthesis or release of histamine. We'll test this later.
The one thing you have done pretty much in common for each experiment is to put everything in your organ bath at once. You do have a control that tests response to histamine alone, but how do you know the leaves aren't having an effect that is independent of histamine? I think there's an experimental control missing. (Your addition of histamine to the ileum bath is really a methodological control, to make sure your organ prep is working right, not an experimental control to help you directly test your hypothesis.)
 
  • #3



Your proposed solution seems logical and well thought out. However, there are a few additional considerations to keep in mind. First, when testing for potential receptor blockade or degradation, it may be helpful to use multiple histamine agonists to ensure that the results are not specific to one agonist. Additionally, it may be beneficial to use a positive control, such as a known antihistamine, to compare the effects of the leaves.

Furthermore, it may also be useful to test the leaves on other cell lines or tissues that also respond to histamine, such as human mast cells, to determine if the potential mechanism of action is specific to dog mast cells. Additionally, it may be helpful to test the leaves on other types of receptors, such as muscarinic or adrenergic receptors, to see if there are any non-histamine related effects.

Lastly, it may be beneficial to investigate the chemical composition of the leaves and compare it to known antihistamines to determine if there are any similarities or differences that may provide insight into the potential mechanism of action.

Overall, your proposed solution is a good starting point, but it may be helpful to consider these additional factors in order to further explore the potential mechanism underlying the therapeutic effects of the leaves.
 

1. What is the purpose of using guinea-pig ileum and dog mast cells in an organ bath experiment?

The purpose of using guinea-pig ileum and dog mast cells in an organ bath experiment is to study the effects of different drugs or stimuli on these specific tissues. The guinea-pig ileum is commonly used as a model for smooth muscle contraction, while dog mast cells are used to study the release of histamine and other mediators involved in allergic reactions.

2. How do you set up an organ bath experiment with guinea-pig ileum and dog mast cells?

To set up an organ bath experiment, the guinea-pig ileum and dog mast cells are first isolated and placed in separate chambers filled with physiological saline solution. The tissues are then attached to a force transducer and connected to a recording system to measure changes in tension. The chambers are continuously aerated and maintained at a constant temperature and pH to mimic physiological conditions.

3. What is the importance of maintaining proper tissue viability in an organ bath experiment?

Maintaining proper tissue viability is crucial in an organ bath experiment as it ensures that the results obtained are accurate and reproducible. This includes maintaining the right temperature, pH, and oxygenation levels, as well as avoiding any damage or trauma to the tissues during preparation and handling.

4. What are some common challenges faced when conducting an organ bath experiment with guinea-pig ileum and dog mast cells?

Some common challenges faced when conducting an organ bath experiment with guinea-pig ileum and dog mast cells include variability in tissue responses, difficulty in isolating and preparing the tissues, and the need for specialized equipment and expertise. Additionally, factors such as animal age, sex, and strain can also affect the results obtained.

5. How can the results from an organ bath experiment with guinea-pig ileum and dog mast cells be interpreted?

The results from an organ bath experiment with guinea-pig ileum and dog mast cells can be interpreted by comparing the responses of the tissues to different drugs or stimuli and analyzing the dose-response curves. Other factors such as tissue viability, control responses, and known mechanisms of action can also help in interpreting the results. It is also important to consider the limitations of the experiment and potential confounding factors.

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