Hi,
I have some problems with carbon tetrachloride (CCl4) -induced mouse model of liver fibrosis, and would like to ask is any of you has experience in troubleshooting this model.
I run model on male Balb/cAnNCrl mice (Charles River origin), dose of CCl4 – 0,5 ml/kg, IP injections - formulation is 5% CCl4 (from Sigma) in olive oil (from Sigma) at IP dose-volume 10 ml/kg, injections twice weekly, for 8 weeks. Controls – injections of olive oil alone
The first study (run in summer) was ok – mortality - 2 /85 animals - this study is ok.
Second study (run recently, so winter) – the same dose – mortality 80% in the first week (controls with olive oil alone are OK).
Therefore I have run additional pilot studies with lower doses, but still, mortality reaches 60 % even at dose 0,05 ml/kg, and there is no visible dose-related loss in body weights or mortality (although animals that survived the first week are very healthy).
I have no clue what could be a problem, why I see such differences between studies in mortality and mice health, even when Im using the same bottle of CCl4 and the same product of olive oils (though different bottle, newly ordered) – I have excluded dose calculations mistakes (triple check with colleagues in lab), IP injection techniques, mice handling…
Has anyone encounter such problems with this model, or have any idea what may be the problem? Is it possible that CCl4 became more toxic, or broke-down to toxic product? This is a simple compound. I thought even that there is some influence of yearly seasons or moon phases :/ so Im am a bit desperate to solve this
I would really appreciate your input and help?
products
michal
Hi,
tou say you use the same bottle, did you check the expiration date of your lot? Could it be that you are using the same syringe for multiple animals and therefore spreading a disease/virus?
Dear Michael,
It may be due to fasting condition of animals before and after injection. Also, animals weight as well as age could affect their response to toxins such as ccl4. Surprisingly, aged animals usually are more tolerant to toxins than young. Please check these two points.
Good luck with your research,
Hi,
thank you for responses.
Answering - olive oil is newly ordered, CCl4 is maybe a 1.5-2 year old but I have no information of expiration date (it was kept in RT in dark bottle) - still all available information sheets say that is a stable compound.
Only one thing prompted me to consider - this particular product (Sigma # 270652) is for HPLC, with very high purity, but here Sigma did not mention anything regarding its usage in animal models (in contrary to other CCl4, with lower purity: # 289116) - is it possible that HPLC-grade compound has smaller but more toxic impurities? Anyway I will order a new bottle of the latter CCl4
I dont think there is any involvement of disease, worsening of condition of animals was apparently random, still thank you for your advice, I will keep that in mind
Mice were not fasted before injection of CCl4, moreover, due to some publications researching influence of melatonin, and daily fluctuation of levels of CYP2E1, on toxicity of CCl4, all injections were in the morning.
At the start of the study, mice were in a very good condition, about 8-10 week of age, about 25 g - no worrying issues observed
Thanks anyway
check the weight variance of your animal and the the first respondent into consideration
Hi Michal Mlacki ,
I hope you have been selected your dose on the basis of the LD50 of your test compound. If not so please do it accordingly by going through the literature.
Thank you,
Good luck
Dear Michael, I've seen the same problem with liver Transplantation. Animals survive in winter but not in summer after surgical procedure. If you find the matter with your experiment m, please let me know.
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