Anyone has experience using IB4-Saporin from ATS to ablate non peptidergic afferents? We’ve started a set of experiments with this using non conjugated saporin as control. Surprisingly, some animals from both groups are developing mobility problems and we have to sacrifice them before we can test anything (they start crawling, they develop rigidity, immobility…). The doses that we’ve used are in the rank of doses reported in papers (we are using 2ug/animal for IB4-Sap and 0.8ug/animal for Sap).
We are injecting intrathecally through the L5-6 vertebral space. Animals are WT C57 male 7wo. Animals injected with saline are perfectly fine, so we asume that the problem is with the saporin.
It feels like the inocuous range of action of the saporin is very narrow and that the specificity of the conjugated molecule is not that specific. The company claims that the saporin is safe at low doses, but they also say that there’s a lot batch to batch of product variation and that you have to test the proper dose with every vial. We are going reduce the dose by half with the next animals, but the results with the doses we have used indicate that both molecules are clearly killing cells indiscriminately, and the doses are quite low for what I’ve read… So I wanted to ask for any advice before we test it again, to know if we must reduce the dose even more or what
Thanks a lot for you help!
I forgot to say: I’m aware that IB4 has affinity not only for the non-peptidergic afferents but also for blood vessels in the spinal cord. I was expecting some motor symptoms in the group treated with IB4-sap because of some level of injury to the blood vessels, but what has surprised me the most is the affection of animals treated only with saporin.
I have not yet examined the histology of sacrificed animals to see what’s exactly being killed, but I suspect generalized cell damage.
I’m interested to hear what others say. I’ve never used.
@dmolliver @liz @tberta @thicunha
Patricia, your posts here highlight a common occurence in our field (and probably others too) - often when you try to apply methods from papers, you find surprising challenges and outcomes that make you realize that a lot of the details go unsaid in the publication. So thank you for bringing the channeleges you’re facing out into the open so we can discuss and learn together.
We have used the IB4-saporin (from ATS) conjugated long ago. The doses are similar to Patricia’s doses. We did not evaluate these animals right after the injections but weeks after (4 weeks normally). We never saw mouse death, and they look like ok.
Thank you for your kind words and support. I feel that I’m always posting problems in the forum but in my previous entry I had such interesting replies that I wanted to try again with this new saporin problem. I also think that sharing the troubles that never reach the papers between colleages can lead to a better practice and not hiting the same obstacles that others encountered before.
I hope that at some point someone will post an entry in the forum in which I can be of any help for a change!
I just realized that your paper is one that I consulted while planning the experiment (Pinto et al. 2019), so you already helped me a lot :). That’s why I was surprised seeing that our injections (2ug per animal of Ib4-Sap and 0.8 per animal of Sap, which are inside the range you tested) are having this poor results for the health of our animals. I’ve checked and rechecked the calculation of the doses we made and they are correct, so I’m wondering if our lot of saporin is specially effective.
When you say that you didn’t evaluate the animals until 4w you mean that you didn’t check if they were developing any problem the days following the injections? I would like to know if you checked their general health because I don’t know if I’m overreacting and puting down the animals for symptoms that would resolve themselves with a little time or some sort of medication.
Also, in your paper you also had TRPV1+ loss with the IB4-Saporin group and a reduction in the response to capsaicin. I know that there are some subsets of non-peptidergic afferents that express the receptor, but I thought that they were a little fraction of them and you report a loss of ~40% of the signal if I don’t recall badly. I was wondering if you tested the specificity of the cell death with the IB4-Sap (not just nociceptors but other cell types) in the DRG and spinal cord. I’m sorry if the question is too obvious but I want to know to which extent this tool is really specific.
Thank you so much!