We are facing a problem in induction of hind limb ischemia in SCID mice. We tried common femoral artery ligation (only knot) and also cut, but it is not inducing ischemia as expected. The limb is totally functional, no difference in the muscle mass and the histo-pathology also showed negative results.
Interesting thing here is, when we tried the same surgical methods in BALB C mice, which is the background strain for the SCID mice, the surgery worked perfectly well and the limb lost its function completely. Histo-pathology results were also positive in BALB mice.
So can anyone suggest the best surgical method for inducing hind limb ischemia in SCID mice? Please cite any references.
We have odne in nude mice and it worked very well. How long do you wait after the surgery to look for a phenotype?
I dont know about scid problem however, you may try and check under laser doppler one hour after surgery,
Also, try to apply two knots at proximal femoral artery and two knots closer to distal end of femoral artery closer to knee and which is proximal to popliteal artery.
I guess that u can't get the typical response after injury because you are in a immunocompromised background...many recent papers are claiming that after ischemic injury immune cells infiltrate the area after ligation and are the real cause of injury. This has been demonstrated in brain ischemia (see http://www.nature.com/nm/journal/v18/n6/full/nm.2749.html). Maybe u can try to ligate the femoral artery in a immunocompetent mouse and then, when injury is present, try to deplete mouse immune system pharmacologically...
However, i think that the absence of histo-pathological hallmarks in the nude mouse is a quite remarkable result itself :-) Is it reproducible??
I did it on SCID mice and never had problems with that method...but I used to do 2 knots and then check under a laser doppler.
Can I ask you how you anesthesize the mice ??
For what it's worth, during eye development in nudes vessels often fail to regress in the cornea. If something similar is happening in the limb, then the tissue may have so many preexisting collatorals that you don't see much perfusion deficit. Have you measured perfusion (e.g. with doppler) to see whether you are getting a reduction in flow?
You have to be very careful where you place the ligatures for the femoral artery ligation model. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2660231/ and http://www.ncbi.nlm.nih.gov/pubmed/16051277 (do a good job detailing how differential ligation schemes lead to differential necrosis). In our experience, we have also seen that if the ligation occurs distal to the popliteal artery bifurcation (which dives into the tissue and not easily seen and can vary across mice) you result in non-ischemia in the distal hindlimb (http://www.ncbi.nlm.nih.gov/pubmed/19777360). A third issue that only comes with continued practice with the model is involvement of the femoral nerve. Any damage to the nerve can have strong effects on distal perfusion recovery and limb use (this probably can also occur without substantial ischemia). In the switch/increased practice between groups, you may have damaged the nerves more with the initial Balbc data that was not occuring to the same extent in the later groups.
With this model in particular, I would only perform experiment groups in tandem to account for increases in surgical skill, especially early on, and have no variation in the surgeon that is performing the ligation. There is high surgeon to surgeon variability.
Different strain should be got different ischemia result by same surgery.
If you understand the anotomy in hind limb of mice, you can design 5 surgerys at less.
all of nerve, Doppler, ligation knotes, are not importent. design more serious model by ligate defferent arteries.
Surgery have to match animal strain.
I want to make one thing clear..this is not nude mice..it is SCID mice.
One major problem here is we do not have a laser doppler setup anywhere close..so the only read out we have is the visible limb function deprivation, gross pathology and histopathology at the end of 7th and 14th day.
@Myriyam- we sacrificed the animals at the end of 7th and 14th days.
I had done some ligation surgery inducing hind limb ischemia on nude mice. The location could be identified upt to external iliac artery from inguinal incision. I recommended that you might try such a proximal location to see if the ligation works. Collateral supply to distal limb in SCID model could not be ruled out according to your description.
Juliane Unger
This is neither a SCID nor a mouse specific observation. In large animals, ligature of the femoral artey is of course leading to a reduced perfusion in the distal area but there is no critical ischemia. Even occlusion of the infrarenal aortic artery can be covered by pigs and most likely also various other mammals (Pig specific vascular anatomy allows acute infrarenal aortic occlusion without hind limb ischemia and stepwise occlusion without clinical signs. Haacke N, Unger JK, Haidenhein C, Russ M, Hiebl B, Niehues SM. Clin Hemorheol Microcirc. 2011;48(1):173-85).
Dear Mr. Grunewald and Ms. Elganfoud. If possible can you please mail me the protocols that you follow for hind limb ischemia on nude and SCID mice respectively at [email protected].
Thank you.
Hello,
Check this study in which I made the "in vivo" experiments, the protocol is in. But it sounds strange to me that in Balb/c the surgery works and not in SCID because we used the same mice for our experiments. Are you sure that all the protocol that you use for both is the same ??
Hi!
Did you ever try to perform the proximal ligation site higher up (more proximal), in the area of iliac artery, and when performing the distal ligatures closing all side branches and/or closing the femoral vein together with the artery? These procedures increas all the injury severity. It has been shown that occlusion of the femoral vein together with fem.artery very probably results in the loss of the injured limb, which is usually not the desired result, as the level of ischemic severity usually is aimed to be severe but curable. Obstruction of the vein is often not recommended due to that, and the fact that in human arterial diseases, the ischemia is based on occlusion of an artery, not vein.
Also, dissecting the vessel(s) between the ligatures produces more grave injury since a single cut has been shown to be less effective due to fast growth of collaterals after the cut (You can find excellent information and advice for the mouse hindlimb ischemia from e.g. jove.com)
I'm sure all this you already know, but I have noticed that the ischemia severity can be increased by simply closing and dissecting more circulatory routes, by widening the dissected vessel area and including the vein in it.
Have a nice summer!
Best regards,
Annamari
My lab is using balbc and SCID mice with femoral artery ligation. We are seeing a much faster recovery with the SCID mice by day 3 than then balbc mice, therefore by day 7 it is explainable why you are seeing no difference between your SCID and balbc due to the fast recovery rate. These results are being checked through dopler scans The surgery is working the rate of recovery is different.
Berry
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