Hydrodynamic gene delivery
The text on this page is taken from an informal compilation of opinions of contributors to the online VOLE List. As such, they are not peer reviewed and may contain differences of opinion. Those wishing to contact the list may contact Adrian Smith.
We've been doing this procedure for a while under Isoflurane anaesthesia, but we are observing some cases of mortality 24h after the procedure. Briefly, mice are injected intravenously into the tail vein with the solution while under Isoflurane anaesthesia. The volume injected is 10% of the animal's body weight, and the whole volume is injected during 10 seconds maximum. After administration we keep the animals in a recovery chamber for a few hours, and in some cases overnight, depending on how they look like. The animals wake up in a state of 'shock', but they recover after a while.
We don't know what the cause of those deaths is and we are looking for ways to reduce them.
The PI has consulted with some collaborators in other countries and it seems like they are doing this procedure without anaesthesia and they don't have incidences.
I would like to know...
- are you doing this procedure with/without anaesthesia?
And your opinion on ...
- whether you think that Isoflurane could be the reason of the mortality we are observing
- whether you think that this procedure can be done without anaesthesia
We've worked on this procedure with a couple of groups:
Yes, definitely do this technique under anaesthesia - and provide analgesia too.
The large volume of injection causes temporary volume overload on the heart - basically right-sided heart failure - and the liver swells enormously, which is painful. It's inhumane to do this procedure to a conscious mouse.
We also pain-scored the mice post procedure, as they looked very 'flat' and the Mouse Grimace Score scored 1-2 30-60 mins later, so we added buprenorphine (prior to anaesthesia), which seems to help their recovery.
We haven't had any mice die as long as 24 hours post procedure but during the initial development, one mouse died under anaesthesia and another within an hour of the injection - at post morten there was fluid in the lungs (as well as massively swollen liver extending nearly to the umbilicus) so it's likely that the pressure caused lung oedema. It didn't seem to be related to isofluorane anaesthesia, as this was used for all the mice.
The lung oedema seemed to be related to injecting slightly smaller mice (there was a minimum injection volume of 2mL and these were GA BL/6's) but now, larger, well-grown mice are chosen if possible, and the volume adjusted to individual bodyweight (up to 10%).
Take care if you are warming the mice before the procedure, as it can result in a slightly heat-stressed mouse undergoing GA and injection. Keep warming to minimum, and/or use local warming of the tail while the mouse is anaesthetised.
When updating our procedure for blood sampling I discovered that the blood volume for a mouse is in average 58,5 ml/kg mouse when looking at NC3R's homepage but according to Karl-Heinz Diehl et al. (2001) A Good Practice Guide to the Administration of Substances and Removal of Blood, Including Routes and Volumes the recommended mean blood volume of mice is 72 ml/kg (63-80 ml/kg). This is quite a difference. Does any of you know the correct answer? Or maybe it just depends of the strain...
There is a difference in rats in the amount of fluid per kg that males and females can tolerate by i/v injection (don't have the figures handy, but females is lower from memory). Obviously mice aren't rats, but I would be cautious in case the tolerance is different between sexes if using both.
