526 replies to this topic

[AgeVivo]

Dear All,

Following thoughts on MPrize at home I had 3 mice at home. Following the buckyball study I contacted the authors of that incredible paper (rats lived up to 54 months, with errors in the lifespan curves in the paper, followed by long discussions) to know if I could test it at home on my mice, ratger than letting they die from aging without any scientific impact. They were very sympathetic; they'd like some independent researcher to reproduce their experiment in a robust and standard way but in the meantime... here I am:

TODO: improve text of this post.

[AgeVivo]

Handling mice at home
Note: this is only what I do, not necessarily what one should do. I've handled mice in labs but it is quite different. I've bought a mini book to know how to handle mice at home, I've asked people selling mice, but then I made the methods slightly evolve at home while testing what the mice seemed to like.

Basic materials:
- a cage and a big water bottle, some bedding and some simple solid toys for them to play
 cage&water.png   263.16KB   44 downloads
- one small ball for each mouse.
 ball-box.png   263.11KB   43 downloads  ball-opened.png   276.3KB   41 downloads

I renew the cage every week or other week (exceptionnally a 3 week interval, in case of long vacations only):
- I put them in balls and put the balls in the sink
 ball-mouse.png   279.08KB   40 downloads  balls-sink.png   248.05KB   31 downloads
note: to hold mice I gently grab them by the tail; not the extremity of the tail
- I clean the cage and toys, put some bedding that absorbs odours (about 2 cm) otherwise it smells within a week, then add some hay on the top (about 2 cm) because they like it very much
 bedding.png   262KB   35 downloads  hay.png   261.04KB   31 downloads
- I clean the water bottle very well, fill it in with tap water and add 5 to 10 drops of decontaminant for fish water (main content is methylene blue)
 detox-water.png   251.19KB   33 downloads
note: it's my own trick that dates back from playing with chemical properties of methylene blue -- you may find it nice or not. I found that it prevents water left during two weeks in the sun to become stale (to know it I tasted a little)
- I put everything back and put the mice back
 mice-cage.png   236.26KB   43 downloads  mice-cage-top.png   237.71KB   46 downloads
The mice are always very curious and when they enter the "new" cage they check everything and are very active. Sometimes like here they even walk upside down, clung to the top of the cage! It is happening less and less however, as they are heavier and older now.

Then I always add food on a side of the cage... but not today! Indeed I am making them hungry for tomorrow to ensure that they will eat a new food...

[niner]

How will you dose them? Oral gavage sounds difficult. I don't think the rats like it, and the paper said something about them being known to not do well with it. Mice are smaller and less robust, so I'd expect it to be more difficult. Are you thinking of just adding it to their food? If they are willing to eat it, I don't see a problem with that, though you may have a tough time matching the dose that Baati used. (scaled appropriately, of course) Do you know the age and genotype of your mice? If so, you could see if they live a lot longer than they are "supposed to". Otherwise, it would be nice to have a control group. How hard do you think it would be (and how costly) to set up a similar experiment with more mice; maybe 30 to 60? (starts to sound like a full time job...)

[AgeVivo]

C60 -- fullerenes -- buckyballs

The authors of the study prepared the treatment and took this nice picture: olive oil on the left, mixed with C60 during two feeks in the center, filtered on the right (5 ml each)
 C60oo.jpg   38.62KB   73 downloads

It must be conserved in the dark so I put them in aluminium sheets, in a cupboard.
Here is the olive oil and olive oil+C60 at home:
 Photo de moi 12.png   250.54KB   64 downloads

In rats they used 0.2 ml/100 g. The bioavailability has not been tested in mice, but basically it is a question of membranes (oil) so it shoudln't be very different. My mice are 35-40g so with the same ratio I should give 0.08 ml a day (the first week). But I prefer to give slightly more because 1) it is in food and not by gavage 2) I have recently read that comparisons across species tend to be better by body surface area rather than by weight (not sure if there were references and not sure why) 3) acute immense doses where found to be non toxic in rats so there is some margin.

Consequence: As discussed with the authors, I decided to give 2 drops per day. On bread or on cheese, depending on what they eat. One drop can be 50 microliters, more or less depending on viscosity and other parameters. I used the end of a plastic baby spoon that has a smooth round shape to create the drops. By looking at the drop, it seems like water (and really not like honey).
 C60-tools.png   244.04KB   46 downloads  C60-spoon.png   240.86KB   45 downloads
 C60-drop-test.png   249.91KB   40 downloads  C60-bread-yumi.png   240.75KB   38 downloads

I did the same with cheese. Putting all in a cupboard. We'll see tomorrow if the mice like it.

[xEva]

Way to go!

Is there a trick to see the pictures? I get "Sorry, you don't have permission for that!"

[jg42122]

Way to go!

Is there a trick to see the pictures? I get "Sorry, you don't have permission for that!"

]
I got the same thing.

[niner]

Fantastically Great Job!

The pictures are attachments. Are they only visible to members? That seems wrong... (I can see them, but that might be mod super-powers...)

[AgeVivo]

Thank you. I will take time to add information about the mice; I may put the images on the web unless someone has a better solution (like visible only from people logged in longecity.org)(I have used the "More Reply Options" button when filling in the post and then attached images).

Giving C60+oil

Cheese = bad. This morning, the C60+oil drops had leaked over and on the plate, next to the small pieces of cheese. So cheese is not a good solution (at least gouda cheese) and so I have to work with bread.

1Mouse per ball to eat separately = bad. I have put a mouse in a ball where I had preliminary put a piece of bread with C60+oil. But the mouse was then too focused on going out/rolling to eat the bread. After 3 minutes I've put the mouse and the ball back in the cage. Perhaps I should train them to eat in balls

All 3 pieces of bread in cage = not equal because they fight for it. I gave 3 pieces of bread directly in the cage. At the beginning it was perfect: they all took one piece and went to different places (two corners and one "house") to eat away from their sisters. But when they were approx half-way, a mice in a corner put her piece of bread on the floor, went to see the mouse in a corner and took part of her piece of bread; she ate it and went to see the third mice in the house and they were both tying to eat the third piece of bread. I couldn't see well what happen because it was in the house but the moving mouse when away and went back to her initial piece of bread finished it, and then all 3 mice went back to the house (where they sleep).
 cage-mice-bread.png   309.78KB   31 downloads
Note: I haven't seen any behaviour that would tell me that there is something special in C60+oil. They started eating the part of the bread that had C60+oil but it is probably because it was on the top, or perhaps because it had some odour for them. They behaved normally. Typically I wouldn't attribute the aggressive behariour to get the bread of others to C60+oil, because once upon a time I give them things that they like very much (curlies) and they they behave similarly. Also going back to sleep is completely normal.

Edited by AgeVivo, 02 June 2012 - 09:41 AM.

[AgeVivo]

Of course I should have trained them to eat in balls before. I haven't, my bad (I thought it would be Ok but no). Perhaps also today I should have waited more than 3 minutes (the mouse was obviously hungry and stressed so I didn't want to wait longer this morning). Anyway, it is the only way I see so I decided to train them now; better late than never.

Training them to eat separately (in their balls)

I have put them in balls in the sink, with their normal food in the balls (usually I put the food directly in the cage). Just like the one tested with C60+oil this morning they were much more busy trying to roll or go out than to eat. After 5 minutes they started to lick (sign that they have been stressed; I know this sign from labs) and after 10-15 minutes they started to eat (alternating between licking, looking to go out and and eating). After 25 minutes they were calm and eating from time to time. Aftert 40 minutes I have put them back in the cage, not sure that they ate as much as they would normally do.
 sink-mice-food.png   312.72KB   33 downloads
My poor little mice. I hope they'll get used to it. I am wondering if it is the best way. Sure, we know that they'll each eat the same portion. But it is stressful (for now at least) and it a lot of time for me (will I find this time every morning of the coming week? I need to put them back in the cage then) and they don't have access to water while they eat. I have some tubes where only one mouse can go, I am wondering if I were to put 3 tubes, each containing C60+oil+bread, if one mouse would still be able to take others' food. Perhaps I can also put one mouse at a time in the cage with a piece of bread; but then it is going to take hours for me in the morning... Or, I leave the normal food in the cage as always, but I put C60+olive oil in curlys, in tubes. Yes I think that's the best. I have the day to think more about it.

Edited by AgeVivo, 02 June 2012 - 09:55 AM.

[niner]

Could you place temporary partitions in the cage to keep them separated until they're done eating? You'd want them to all have access to water, I guess. It sounds like to do the experiment right, you'd need two more cages and water bottles. I'm sure Longecity could pick up the tab on that... One possibility would be to put two of them in balls, and feed the third in the cage, then swap them. That's more work and time, though.

[AgeVivo]

Thank you - yes/perhaps: the thing is that whenever you modifify their environment they spend much time investigating the new environment and do not think of eating during that time. So I'd need to put the split for some time, let them get used to it, and then add curlies; issue: I don't want them to eat the split or go over and I don't know how to make such a split (in very hard plastic or in metal). Swapping them would indeed work but I hope to find a less time consuming protocol. 3 individual cages would go but it would be only slightly better than eating in the 3 balls (I don't want to have one mouse per cage during the whole experiment: my mice like being together).

This afternoon I looked for such a solution based on curlies where I don't have to starve them. (it would be much easier for me and much more normal for the mice). Indeed, when I put some curlies in the cage, they come and eat them; they also fight a little for it, but generally less than this morning because they are not hungry and because I put more than 3 curlies.

So I gave them 3 half curlies in their cage, they went to 3 different places to eat them, and when most of it was eaten they started fighting and one mouse abandonned its part and tried to take the part of others, etc (it is not the same mouse as this morning who globally won). Amost perfect but not exactly, because of scarcity of resources. I guess that if I give them 3 quarters-of-curlies, and that they are not hungry, it should be OK because they will have eaten it before the fight starts.

A little later I have then put the tubes in the cage, with 3 quarter-of-curlies. They ate very very little of the curlies but spent much time on the tubes, because this was the novelty (although they used to know the tubes). So I removed the curlies and a tube, and let them get used to the last tube. Later I put back the quarter-of-curlies in the tube (and one outside), but they directly removed them from the tube and started fighting around without thinking of the tubes as protected places.
 curlies-tubes.png   257.21KB   25 downloads

I guess (/hope) I'm not far. Some time ago I had scotched the tubes to the top, so that it was a bit dificult for the mice to get in and out, and it worked as protecting places. So I'm going to try it, or simply the quarters-of-curlies without the tubes... I hope to get a working protocol today.

Edited by AgeVivo, 02 June 2012 - 04:05 PM.

[zorba990]

I have a question. Do the mice in these studies ever procreate? Just curious about testing C60 with respect to that -- something that might be important to humans considering taking it.

[AgeVivo]

Tadaa! It seems to work. Well, N=1

I have attached the tubes to the top of the cage. When I put the quarters-of-curlies in it, it is slighly difficult for me so I naturally shake a little the cage, and they get interested in what is going on and wake up. When I close the top, they want to see what happened so they go to see the tubes. Once a mouse finds a piece of curly, it stops to eat it (without going outside of it, because going away from the tube is not easy, so they first eat). Eating takes some time; during that time all mice have found a piece of curly, in a tube. Once a mouse has finished its curly, it looks for other places and would like to steal others' food, but it is not possible because they are already in and protected by the tube.
 cage-mice-tubes-top.png   309.82KB   21 downloads
At least that's what I hoped and what I've seen. It should work with more mice as well (perhaps with half a curly actually, to give more time). I hope it'll work next times as well. The only thing is that they must be fit to climb up the tubes(it doesn't work for ill mice), but since the treatment is not to be taken during the whole remaining life, that's ok.

Edited by AgeVivo, 02 June 2012 - 06:46 PM.

[AgeVivo]

N=2, but a little by chance (none of the mice had found that there was a third piece; it is only after a few minutes that the mouse who had not eaten curly discovered it)

So tomorrow morning i'll swap mice, as you suggested niner, to ensure that they all get their dose of C60+oil, and during the day I'll train them from time to time to know that there are 3 curlies, so that if it works then next mornings it will be easier.

Best, AV.

Edited by AgeVivo, 02 June 2012 - 09:06 PM.

[AgeVivo]

2nd feeding: I made it but it was long and hectic: hey mice, are you teasing me with food when not hungry??

this morning I have prepared quarters-of-curlies + C60+oilive oil.
 3-quater-curly-oo.png   256.26KB   16 downloads

Mouse 1. I have then put 2 mice away from the cage (in balls, in the sink), and put a quarter-curly-oo in the cage. The mouse only ate very very little piece of it, with no C60oo... I waited for 15 minutes but it did not change. Perhaps the mouse was slightly scared because it was alone in the cage, I don't know. So I prepared bread+C60+oo, and she took it! Must surprising, she actually ate half of it, and it is the half that contains C60+oilive oil!! I put her in a ball (separate place),

Mouse 2. , and put another mouse in the cage. Put a quarter-curly--oo and she ate it. great. I put her in a ball (separate place),

Mouse 3. and put the last mouse in the cage. It licked once the olive oil+C60 on it (took about half of it) and then abandonned the curly. To test if it was because of the C60oo, I gave a quarter-of-curly without C60+oo, but the mouse did not take it. I prepared then bread+C60oo with only one drop, it took a small part of it, including a little of the C60oo. Not the extact dose so far, but not far.


Interpretation?
Pff... yesterday they were taking every piece of curly. Is that the one too much after one night? Is the olive oil+C60 + curly association strange to you? (the olive oil+C60 seems good when put on bread). Where you not at ease to be alone in the cage? To test the later I've put the 3 mice in the cage, with the remaining food (3 pieces of curly (one without C60oo) and 2 pieces of bread (one without C60oo). Mouse 2 ate a portion of the almost-not-touched curly from mouse one -- so she got a little more than the decided C60oo overall -- and mouse 3 took the rest of it. The rest remain untouched and they went to sleep.

Conclusion
2 mice took the right dose, one took at little more. It took me almost 2 hours total, and I have not been able to infer any clear method for the swapping method in non starved animals.

I guess I have no choice for the next days: Algorithm:
- either today I am able to robustify yesterday's tube method without starving mice
- or I make them hungry every day during a week, then every other day etc, and in the morning I swap them for C60+olive oil on bread, then give them food. And in case they seem not to want to eat enough outside of the cage I may have to put the food in the cage and change the cage every evening (every other evening etc) to remove food (!!!)

[Turnbuckle]

I have a question. Do the mice in these studies ever procreate? Just curious about testing C60 with respect to that -- something that might be important to humans considering taking it.

Here's a relevant study: http://www.ncbi.nlm....pubmed/20063269

They used radioactively tagged C60 and found that C60 does cross the placenta and does show up in milk, but they didn't mention any abnormalities as a result--at least not in the abstract.

Edited by Turnbuckle, 03 June 2012 - 11:17 AM.

[AgeVivo]

I tried the tubes method again and now it doesn't work anymore. I guess it is not new enough to be interesting for them:
One mouse climbed in, ate the piece of curly, ate it. The two others smelled and did not go up. I then did it with bread, again one mouse climbed (not the same one and not the same tube) and two others did not. I understand that yesterday it worked because of novelty and "competition" among them (and they eat their trophy), and that probably they have had too much of the smell of curly around yesterday and today so it is not so interesting anymore.

So I'm going to remove the tubes and continue just as I did the first day: having them starve overnight, eat in the morning (c60oo one by one, then food in a big ball where all three will be (I hope it will work) then put them back in the cage before going to work, feed them again when I come back from work, etc).

Edited by AgeVivo, 03 June 2012 - 12:18 PM.

[AgeVivo]

Analyzing the mice, during the day one mouse was not at its best this morning/noon (the white one; hair not very smooth, wanted to sleep) but now it is active and has a nice hair. Nothing to worry about, it happens from time to time, and it may be due to all the things I did around. The two other mice (black, they look very much the same) are (and have always been) in a very good shape.

[Raphy]

Hello AgeVivo,

Wouldn't starving the mouse overnight increase their lifespan by intermittent fasting? Or is 12hours of "fasting" no enough to be relevant in this matter?

[Godot]

It seems like methylene blue in the water could be a serious confounding factor.

[AgeVivo]

3rd feed done

[Raphy]

Hello AgeVivo,

Wouldn't starving the mouse overnight increase their lifespan by intermittent fasting? Or is 12hours of "fasting" no enough to be relevant in this matter?

[tweedlover]

If you want to determine the effect of the solution I think it is absolutely critical not to starve them.

This paper found that by only giving access to food 8 hours a day (and same calories) lifespan was increased in mice by 40% as well as improved liver function, reduced inflammation etc. There is a strong possibility that this is the effect that was observed in the fullerene study as well since the study admits that the mice were starved. One of the most important things your study could do is rule out that possibility by giving them more access to food - but would require a bit more work to get your feeding method right.

http://www.cell.com/...550413112001891

[tweedlover]

Another angle - I am not a scientist, but this 2008 study appears to show a lifespan increase of 11% and increased brain function from dosage of some kind of Fullerene SOD Mimetic...

http://www.owndoc.co...pan-of-mice.pdf

[Turnbuckle]

It seems like methylene blue in the water could be a serious confounding factor.

I agree.

[AgeVivo]

Answer to questions above:

With usual mild potential lifespan improvements you indeed need many mice to detect effects. But here we are in a very special case where C60oo was reported to lead to 50-54 month old rats!! it just happens that I already had 3 aged mice, so however unprecise my conditions are I thought I should try: if only one of my mice reaches such a lifespan then it will be compelling evidence. But clearly as I said many times the best would be that C60oo is tested in a standard rat facility.

I have are bought the mice in a petshop so they are not from a specific known strain. Based on others' at home experience the lifespan is typically 2 - 2.5 years. Godot, Turnbuckle, the last mouse I raised from the same petshop also drank the methylene blue and lived up to such an age (To find: it should be in the mprize thread where I was regularly posting and in my experiment notebook). I saw it age despite initial good shape so it was not accidentally short lived, but the least I can do is admit that it is poor evidence. I'm greatly waiting for the ITP mouse lifespan results with methylene blue.

Raphy, Tweedlover, actually it is just a temporary treatment (two weeks of overnight starvings and then one overnight starving per week during a few weeks). Intermittent fasting is not that powerful and the 40% life extension you mention is a comparison with short-lived obese mice due to ad libitum high fat diet (makes me think of "supersize me"...).

Procreation: Zorba, lifespan experiments do not mix males and females in the same cages. Turnbuckle, interesting

Edited by AgeVivo, 05 June 2012 - 01:22 AM.

[AgeVivo]

Tweedlover indeed there are other papers indicating life extension with fullerenes:

Another angle - I am not a scientist, but this 2008 study appears to show a lifespan increase of 11% and increased brain function from dosage of some kind of Fullerene SOD Mimetic... http://www.owndoc.co...pan-of-mice.pdf

- Indeed, this paper from researchers in the US [1]. Here are the lifespan curves, you can see it was done in a lot more mice ("a" is a part of "b")
 C3-qurvival.png   84.03KB   41 downloads
and just below in the paper you can see that weight and food consumption suggest it is not crypto-CR (true to for the C60oo study).
- The C60oo authors told me about another paper with other fullerenes from authors in China showing mild life extension in rodents. I'm not able to find it now (perhaps someone else here?[2]). They told me they attribute the much greater longevity of C60oo due to the fully optimized structure of C60 to capture free radicals thanks to the numerous double bounds on the buckball structure. But it is just a hypothesis of course.
- While searching for [2] I found another paper [3] from reseachers in the US showing life extension if various species with other fullerenes.

To me this is all nice but the claims/results of the C60oo study are particularly extreme. Therefore what I am doing.
* * *

[1] Kevin L. Quick, Sameh S. Ali, Robert Arch, Chengjie Xiong, David Wozniak, Laura L. Dugan. A carboxyfullerene SOD mimetic improves cognition and extends the lifespan of mice. Neurobiology of Aging 29 (2008) 117–128. Free full text: http://www.owndoc.co...pan-of-mice.pdf
[2] ?
[3] Gao J, Wang Y, Folta KM, Krishna V, Bai W, Indeglia P, Georgieva A, Nakamura H, Koopman B, Moudgil B.

Gao J, Wang Y, Folta KM, Krishna V, Bai W, Indeglia P, Georgieva A, Nakamura H, Koopman B, Moudgil B.

PLoS One. 2011;6(5):e19976. Free full text http://www.ncbi.nlm....25/?tool=pubmed

Edited by AgeVivo, 05 June 2012 - 05:28 AM.

[AgeVivo]

4th feed done. The mice look in ver really good shap but perhaps I am being subjective

[tweedlover]

Hi AgeVivo

Raphy, Tweedlover, actually it is just a temporary treatment (two weeks of overnight starvings and then one overnight starving per week during a few weeks). Intermittent fasting is not that powerful and the 40% life extension you mention is a comparison with short-lived obese mice due to ad libitum high fat diet (makes me think of "supersize me"...).

Good to hear that the intermittent fasting is pretty minimal and agree high fat changes things but my point is that IF has an effect that should be controlled for - in some cases a very marked effect.

 

I'd still say that no fasting would improve the experiment, given that IF is shown to have similar effects, and nether the fullerene nor the IF life-extension mechanisms are well understood.

Alternatively, you could have a control group who were fed at the same times and not given fullerenes.

I'm sure this is coals to newcastle, but here is what Wikipedia has to say on the subject of IF in mice - note the optimal frequency of the main study which increased lifespan by 15-20% being 1 day in 3 - clearly less frequent fasting can also have some impact.

http://en.wikipedia....#Animal_studies


Animal studies

The 1945 study by Carlson and Hoelzel, referenced above, found that the apparent life span of rats in the study was increased by intermittent fasting. Tests in which a group of thirty-three rats were allowed the same food ad libitum and groups of thirty-seven, thirty-seven and thirty rats were fasted 1 day in 4, 3 and 2, respectively, after the age of 42 days, showed that the optimum amount of fasting appeared to be fasting 1 day in 3 and this increased the life span of littermate males about 20% and littermate females about 15%. However, the pre-experimental condition of the individual rats was also found to be an important factor determining the life spans. No drastic retardation of growth was produced by the intermittent fasting but the development of mammary tumors was retarded in proportion to the amount of fasting.^[1]
A number of subsequent studies have shown beneficial effects of IF in animals.

• "Reduced serum glucose and insulin levels and increased resistance of neurons in the brain to excitotoxic stress."^[2]
• "Enhance[s] cardiovascular and brain functions and improve[s] several risk factors for coronary artery disease and stroke including a reduction inblood pressure and increased insulin sensitivity" and that "cardiovascular stress adaptation is improved and heart rate variability is increased in rodents" and that "rodents maintained on an IF regimen exhibit increased resistance of heart and brain cells to ischemic injury in experimental models of myocardial infarction and stroke."^[3]
• May "ameliorate age-related deficits in cognitive function" in mice.^[4]
• Correlation with IF and significantly improved biochemical parameters associated with development of diabetic nephropathy.^[5]
• Resistance in mice to the effects of gamma irradiation.^[6]
• Lifespan increases of 40.4% and 56.6% in C. elegans for alternate day (24 hour) and two-of-each-three day (48 hour) fasting, respectively, as compared to an ad libitum diet.^[7]
• Rats showed markedly improved long-term survival after chronic heart failure via pro-angiogenic, anti-apoptotic and anti-remodeling effects.^[8]

Here also is a study referenced in Wikipedia showing lifespan increases of 40-56% in worms from intermittent fasting (alternative day) without calorie control.
http://www.ncbi.nlm....9?dopt=Abstract

 

[AgeVivo]

The rats of the c60oo study fasted overnight before being treated. I don't think it impacts lifespan much much (the group being treated with water was not super long lived), but in case it does (or in case the temporary combination of overnight fasting and taking oil has) then it is certainly good to reproduce the overnight fasting!

Edited by AgeVivo, 05 June 2012 - 01:59 PM.