LPS lethality - why isn't it working

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angelaevan
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LPS lethality - why isn't it working

I am trying to establish a lethal dose prior to some efficacy testing and I can't get a mouse to die, even getting them to look ill isn't consistent.  I have worked with phenol extracted LPS from Sigma doses up to 900 micrograms per 20 gram mouse and now I added D-galactosamine (hydrochloride) 20mg/mouse with doses up to 400ug/mouse and still no lethality - I am reconstituting the LPS in 0.9% NaCl with lots of vortexing at 10mg/ml.  Any suggestions?

marcus muench
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I'm just in the middle of

I'm just in the middle of writing up an animal protocol for exactly this kind of experiment.  So I'm very interested in your situation.

What kind of LPS are you using?  I've seen both e. coli (either 011:B4 and O55:B5) and  Salmonella abortus LPS used.    Most references I've seen use PBS, but I don't know how important that is.

What strain of mice?  Seems Balb/c are commonly used, but other strains work as well.

Are you injecting the LPS i.v.?  If so are you getting good injections, not just pumping the tail full of LPS?

Your results are puzzling as i seems your mice should by falling over simply at the sight of that much LPS and D-galactosamine.

angelaevan
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I am using 055:B5 LPS the

I am using 055:B5 LPS the phenol extract,  IV tail injections (definateley IV - so far that's the smoothest part).  For mice i went with the less expensive ICR males (approx. 5 weeks old) and I have considered the difference between my outbred strain and the usual inbred strains, yet I find it difficult to convince myself that there is so significant of a difference.  I saw another post on this site from someone that when they reconstitute their LPS they heat it to 50C.  Next try I may try out 37C water/shaker bath for 30minutes and see if that helps.  I spoke with a consultant from Sigma to see if there are any in vitro tests to validate my LPS but no, and they seemed to think the difference between NaCl and PBS was negligible.  I even considered adsorption on the polystyrene cryovials and have changed from 1mg/ml LPS to 10mg/ml and then I take the dilution back down just prior to injections.  What do you think?

marcus muench
marcus muench's picture
 Thanks for the details.  I'm

 Thanks for the details.  I'm trying my connections to see if I can get some more insight into why what your doing isn't working.  Will post when I have more information.

angelaevan
angelaevan's picture
Just thought I would let you

Just thought I would let you know that I have been checking daily for any additional advice.  I have now tried with up to 1600ug/20g mouse or 80mg/kg in the galactosamine sensitized model and I still can only produce mice with fevers.  I am starting to question my resuspension techniques yet I do not find any detailed description in the literature, which I believe would have been included if it was that relevent.

I have even started emailing authors of the literature I am finding.  I have yet to receive any response.

marcus muench
marcus muench's picture
 I haven't been able to find

 I haven't been able to find a paper in which they used ICR mice.  Have you?  I have seen some work that suggest that there are some real differences in sensitivity to LPS among strains of mice, so maybe this is your problem.  Balb/c and C57BL/6 appear to be among the most sensitive.

As for LPS reconstitution and type, I've seen all sorts of methods including what you mentioned.  Usually some sort of sonicator treatment in PBS or saline.  But I have seen using a 1:1 mixture of pure ethanol and water and sonication to get a 500 microgram solution followed by further dilution in media.

Please let us know how things are going, and I'll update if I find out any further tips.

marcus muench
marcus muench's picture
 I did find some papers using

 I did find some papers using ICR mice, but they all seemed to use LPS with another agent combined.   I guess those mice should work, but my inclination would be to try another strain of mice to see if you have any different results.

As I'm still working on writing my animal protocol to get approval fro similar experiments, it is going to take me a little while before I get some first hand experience.  

I'll post more if I come across anything of interest.

angelaevan
angelaevan's picture
Allright - since my last post

Allright - since my last post i have changed 2 variables - First I switched LPS to 0111:B4 - no lethality I tried it at 10ug/mouse and 20ug/mouse w/D-galn sensitization at 20mg/mouse..then...same dosage but I used C3H inbred females - so far I have 75% lethality in both groups and it's only just getting on 24 hours - I'll be repeating it today and I'll let you know
I find it difficult to accept that inbred ($20/mouse) vs. outbred ($4.50/mouse) is the difference.  I always recommended muts as pets and I guess this supports that but, wow.

marcus muench
marcus muench's picture
 I had a chance to ask

 I had a chance to ask someone from CharlesRiver today about this issue, and they also guessed that the outbred strain was just plain tougher.  They were going to pass the question one to others in the company and get back to me.

Either way good to hear your mice are dying (don't get to say that all the time).  

I assume your not using C3H/HeJ, since they are LPS resistant (tlr4 mutation).  

Unless someone convinces me otherwise, I'm leaning towards using Balb/c in my protocol.

angelaevan
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In hindsight I agree and I

In hindsight I agree and I would recommend Balb-c, they are the same price in my neck of the woods and I found the C3H (C3H/Hen-Taconic's) are a little more challenging for IV injections since they are colored.  But now that it's working I don't think we are gonna keep changing any variables.  Now I want to take the dose down to find the lowest lethal dose and maybe go back and try it with the 055:B5 - but my boss (the PI) wants to forge ahead and start checking the efficacy of our plasma - our company hyperimmunizes horses and pheresis's them.  I'll keep you updated - I have been reconstituting in the same container it came in and also sonicating in it for 2 minutes just prior to the mixing with D-Galn and saline and putting it in the syringe - now with the plasma I will need to incubate it in the shaker bath for 30 min. w/plasma we'll see how that goes.  I did the same process with establishing efficacy for an anti-venom product but the USDA did not like the testing method too much, they wanted an LD 50 curve established which doesn't make sense to me since this seems to prove more.  I'll keep you updated.