Mice behavior after Tamoxifen injection: what does it look like?
Dear community, I will be very grateful for any clues about the issue. I was stacked with problems regarding mice's behavior immediately after an intraperitoenal injection of Tamoxifen.
Mice's behavior changes drastically after injection. They become much less active, sometimes with no movement at all; their eyes are squinted; some of them move only a couple of steps even after being touched by the experimenter. One interesting fact is that they sit separately, while normal mice are trying to get together. I observe such conditions even 24 hours after injection. In my previous experiment, I observed the same behavior after injection, and 13 of the 18 mice died. That was so frustrating. In my recent experiment, I used a completely new reagent, and I was extremely careful with the preparation of tamoxifen, including dosage and injection. I have even slightly reduced the injected amount, just by 0.005-0.01 ml. But still, the behavioral changes looked exactly the same. I am really perplexed because MANY articles use this concentration and dosage, and they refer to it as safe! One1 of the articles even declares that "Over several years, we observed a mortality rate of 3.7% after three injections of TAM (n = 4.080, adult"mice)" — they injected 100 mg/ 1 kg for three consecutive days! I saw the articles about the influence of tamoxifen on some behaviors (anxiety-like, depression-like, locomotion, etc.), but I cannot find a detailed description of the behavioral phenotype immediately (during 48 hrs) after injection.
If anyone can share their experience, I will be grateful!
Short information:
- Mice: C57BL/6, 11 weeks old, 26–30 g.
- Purpose: initiate targeted recombination in the active population of neurons in the brain. The cocktail of viruses was delivered to the brain; after four weeks of recovering tamoxifen was injected
- Tamoxifen: fresh solution was prepared before every injection from tamoxifen powder (Sigma), dissolved in corn oil at 60 degrees for at least 2 hrs, shaked in a shaker every 10 min, covered by foil. The solution was completely transparent before injection.
- Concentration: 20 mg/1 mL of corn oil
- Dosage: 100 mg/ 1 kg of weight, about 2-3 mg per animal
- Injection: peritoneal, under slight isofluran anesthesia to reduce mice movement during injection; with 1 mL surynge and 27 G needle
1) Article Refined protocols of tamoxifen injection for inducible DNA r...
Hi Ilia V. Potapenko,
Do you observe this behaviour immediately after the very first injection, or only after few of them?
The problem with injecting oil IP is that it will stay in the peritoneal cavity, and may cause inflammation (sometimes leading to peritonitis and death). This is likely the cause of mortality when performing tamoxifen IP injection.
I'm also wondering whether the isoflurane anesthesia before IP may interfere with tamoxifen somehow. As you said, many people do IP injection for tamoxifen treatment, and do not have the same issues (at least not as dramatically), but usually, people don't use isoflurane anesthesia prior to IP.
In our hands, we always do tamoxifen treatment by oral gavage, to avoid the aforementioned peritonitis. Our dose is higher than yours: 150 mg/kg for 5 consecutive days, by delivering a small volume of a 50 mg/mL tamoxifen solution in corn oil (90 µL for a 30 g mouse). We use flexible gavage needles to reduce the risk of oesophagus damage if the mouse struggles. Overall, this is pretty well tolerated, even though it can happen that some mice exhibit sickness behaviour after the first days. Very rarely, we had to sacrifice mice that reached an endpoint, and our "mortality rate" is definitely much lower that 3.7%.
Alternatively, if you're not comfortable in using gavage, you could try doing subcutaneous injections instead of IP, but the oil will also remain there.
Dear Vincent Simon ,
Thank you for your answer and suggestions. Unfortunately, we cannot use oral administration.
I think we have finally figured out the reasons.
The first is an old corn oil. Our colleagues told us it may have severe side effects. Postmortem observation of some animals revealed a lot of white fatty formations throughout the mice's viscera. While individual organs looked okay (that is not 100% certain since we are not mice anatomists).
The second is an isofluran anesthesia. You were right; isofluran seems to react somehow with TM. Another side effect is that mice rapidly move when waking up after anesthesia. If it happens during injection, the probability of damaging organs is rising.
The third reason is the size of the needle. Initially, I used a 30G needle. In that case, the injection takes quite a while. It is often enough for mice to start moving, trying to free themselves (even without isofluran). The needle can easily hit some organs as well. When we changed the needle to 27G, injection time was drastically reduced.
And the last reason is the preparation of TM. It should be completely dissolved. It sounds obvious, but it is still so important. A visually transparent solution may still contain small crystals that could be detected by spectroscopy. It is better to wait a little longer, constantly shaking the solution. Now we put TM at 37 °C in a rotating shaker over night.
After these measures, the mortality rate decreased greatly!
I hope it will help anyone!
Dear Ilia V. Potapenko,
Glad that you've solved the problem, and thanks for the detailed report, this is very valuable information.
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