Hi,
I am currently trying to establish a model of intestinal ischaemia/reperfusion injury in mice, in my lab, with a goal to producing local injury and acute lung injury as a result. However, I am currently having issues with mortality in my I/R animals. My protocol is as follows:
1. Anaesthetise male mice (8 weeks old) with ketamine/xylazine i.p. and transfer to a heated platform.
2. Perform midline incision and laparotomy.
3. Identify superior mesenteric artery. I/R animals have the SMA clamped with a non-crushing surgical occluder for 30-60 minutes (have tried both). Sham animals are treated identically, except for clamping. Ischaemia is confirmed by pallor of the intestines and reflex tachycardia.
4. Gauze is soaked in warm PBS to cover the surgical site for the extent of ischaemia.
5. Clamp is removed to reperfuse the intestines and 1mL of warm PBS is administered i.p. before suturing the muscle and skin walls closed.
6. 30 mg/kg Evans Blue is administered to assess inflammatory oedema secondary to injury.
Mice that underwent 60' ischaemia died at 75 and 90 minutes reperfusion, whilst the sham counterparts survived. Mice that underwent 30' ischaemia died at 45 minutes reperfusion, whilst sham counterparts survived.
Clearly, this is an issue related to the severity of the model, yet so many publications use similar ischaemia/reperfusion timepoints - does anyone have any ideas of where I might be going wrong? I could try and reduce the ischaemic time further, but I risk not inducing a severe enough injury to give me the readouts I need.
Any/all input would be greatly appreciated!
Thanks
Ross
Ross, I guess this is inadequate resuscitation. 1ml PBS sounds like a lot but so is fluid loss after total bowel IRI. Try to slowly inject fluid in the spleen (30G needle and then compress with earbuds 30 sec)... that is iv... besides leaving 2-3 ml ip. Then use warm pad or lamp.
Then, Look at the bright side: you have a mortality that will make the effect of an intervention pretty visible.
Good luck!
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