Dear all,
I've been doing some experiments using intranasal dosing of LPS, a technique that has been well described in the literature and I achieve very uniform results (on the whole). Occasionally there will be a mouse that I believe hasn't had the dose as their lungs show very little sign of injury, and no sign of systemic illness. I am loath to discard the results from these mice as it could be that these animals are protected for a specific reason, or it could mean that the dose was swallowed during dosing. Does anyone know of a measurement that could be taken after dosing to ensure that the LPS did reach the lungs? That way I could confidently exclude the tiny minority of mice that I believe didn't receive the full dose.
All the best,
Charly
A successful administration might change the phenotype of peripheral blood leukocytes. I would try a FACS-staining of PBMC "before/after" (maybe at different timepoints 2-12h after) and check for expression of CD69 on subpopulations (T cells, B cells, NK cells). In this paper they show that after airway LPS-exposure an increase of CD69 signal can be measured:
A Ortiz-Stern et al. (2011): Langerin+ dendritic cells are responsible for LPS-induced reactivation of allergen-specific Th2 responses in postasthmatic mice.Mucosal Immunology (2011) 4, 343–353; doi:10.1038/mi.2010.73
The traditional test for endotoxin, the limulus lysate test, could probably be performed on pulmonary secretions or perhaps even on blood, depending on the dose of LPS that is being administered.
Is there some other tag you could put in your LPS prep that is easily detected to serve as a surrogate. Perhaps some fluorescent compound or microbead. I am glad to hear you don't wish to discard the mice from your experiment until you know what is going on. Too many people discard "uninterpretable" results leading to a bias in the analysis.
I'm wondering about that at the moment- there is an albumin conjugated to alexafluor-594 and I was considering mixing a little of that in with the LPS. I just don't know how long it'd persist for and as some of my experiments run over 5 days it could be tricky. If it crossed from the alveolar space into the plasma then I may be able to pick up the fluorescence in the plasma which would be ideal - I just don't know how big a dose I'd need to give in to pick it up in the systemic circulation. With the limulus lysate test does anyone have an idea on the dose needed to get a positive plasma sample? I use 50 micrograms currently which works well without causing any mortality. I'd be very happy to blood sample them after dosing to double check, at least that way I know I can trust my results!
You seem to have two different questions. One, is the initial dose of LPS being delivered to the lungs, and two, are you getting an effect from the dosing. The first question might be answered with a fluorescent tag. There are even quantum dot reagents that might allow external scanning of the mouse to see what made it to the lungs. It has been several years since I did work like this so am not sure of the state of the art these days. The second question will most likely require sampling of blood or tissue looking for some sort of biomarkers (t-cell, macrophage activation, cytokine levels, etc.). I would want to know if albumin will traffic in the body the same as LPS before I make too much of it, even if it was measurable in the blood.
The most important is the delivery to the lungs as its effects once present I'm very confident of. I'd love to do it with quantum dot but we don't have the facilities to do that here. Sampling of blood is what I was looking at, but I don't know what will reliably be expressed even if the mouse has a degree of protection.
Re-challenge the mice showing little or no signs of pumonary injury, and administer the dose via an endotracheal tube to ensure optimal dosing.
But then I risk over dosing and those mice won't be comparable to the others!
You indicate in your initial question that you only seem to see a few mice with no indication of receiving the LPS dose. Just note them and run the rest of your experiments. Then analyze the data with and without the questionable mice. Always show and discuss all the data, but state your reasons for analyzing the data in this fasion. If you run enough mice then a few outliers shouldn't affect things. If you truely have a wide range of effective dosing then that might be a problem. Do you have to dose them in the lungs. Can you just inject peritoneally?
Yeah I think you're right, when I was developing the protocol occasionally I'd have one that didn't seem to respond but retrospectively I think I probably was just less experienced at judging the depth of anaesthesia. I can inject peritoneally but you get a much less severe lung injury and that's the topic of my research. I'm considering a double dose protocol though with ip LPS and subsequent IN dosing.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology