526 replies to this topic

[AgeVivo]

** Minor scheduling adaptation, mentioned for traceability **

The paper says: "Three groups of 6 rats (10 months old, weighing 465  31 g) were administered daily
for one week, then weekly until the end of the second month and then every two weeks until the end of the 7th month".

I started the experiment in the beginning of June. To fully respect the scheduling on the paper I should give them the treatment in two days, then the week after and every other week. Instead, due to my vacation schedule and because I think the adaptation is very small, I will give them the treatment tomorrow morning (decay by one day; small in comparison with one week) and then every other week (decay by one week of when to start "every other week" treatments; small compared to 2 months). The scheduling change is of course very minor compared to the huge differences of what I do compared to the paper (mice versus rats, 18 versus 10 months at start, gavage versus bread, other): I am just mentioning it so that people reading my posts follow what I do.

[AgeVivo]

Mice still very active and looking very healthy. Fed treatment this morning.
 bb210712.JPG   51.83KB   11 downloads  bw210712.JPG   21.22KB   18 downloads  w210712.JPG   72.59KB   10 downloads

[Danail Bulgaria]

AgeVivo, if I understood correct from Your postings, at this moment the mice are taking the c60 for 20 - 21 days. Does the mice show some difference between the control and the working group? I mean are there any signs of general intoxication in the mice of the working group, taking c60?

[d4shing]

General intoxication, fuck yeah!

These are his pets - there aren't any controls or blinding.

[AgeVivo]

indeed this is about the most limited experiment you can imagine. no control nor blinding. it is only interesting to setup methods, check that it is not very toxic, etc. and in case the mice are abnormally healthy for their age (I think that for now there is a good case but nothing conclusive at all) and live very long (if they are still alive in one year then to me it is quite conclusive).

Edited by AgeVivo, 26 July 2012 - 12:52 PM.

[Raphy]

Hey AgeVivo,

I can't find this information anywhere: how old are the mice?

Thanks for your work
Raphael

[AgeVivo]

I have given information on their age previously in the thread. With a precision of about a week, I started when they were 18 months and next week they will have lived 20 months.

Edited by AgeVivo, 26 July 2012 - 11:31 PM.

[Raphy]

Thank you AgeVivo

[AgeVivo]

Hello,

Today I came back from 2 week vacations. Good news the mice are doing well (of course this was expected; it is not the first time that I go on a vacations). Here is a video: http://agevivo.com/l...040812-mice.avi

Although I notice that they are less "sportive". Example with a video: http://agevivo.com/l...40812-sport.avi . Perhaps it is due to aging (scarcopenia, rhumatisms... aging), perhaps it is due to the lack of c60 for 2 weeks (coming back to a more normal state for their age?), perhaps it is due to the fact that I just came back and somehow disrupted their rest/cycle (during vacations I put a 12h day/12h night light in the kitchen); I'll try to keep an eye on their motricity in the coming days.

I have fed them with bread+C60-olive-oil (without starving because I thought that the novelty aspect of bread would be sufficient; more or less: I had to wait long for them to come back and forth to their piece of bread and finish it). I changed the cage. Here are pictures, as always:
 bb040812.JPG   53.24KB   12 downloads  w040812.JPG   76.91KB   9 downloads  bw040812.JPG   37.19KB   5 downloads

I am wondering if I should give them C60 next week-end or the week-end after. With the plan I scheduled planned it should be the later. But in case the reduced activity is confirmed during the week, I'd like to have a feeling of whether it is due to spaced c60 feedings.

[Logic]

IMHO feed em whatever it takes to keep em young and alive as long you can.
Why repeat an experiment thats already been done?

[tintinet]

IMHO feed em whatever it takes to keep em young and alive as long you can.
Why repeat an experiment thats already been done?

D'ya ever hear of cold fusion?

[Logic]

D'ya ever hear of cold fusion?

Good point.
But what would have happened if the Baati rats kept getting C60oo?
Perhaps an even better result?
They seem to have been too keen to end the experiment from my reading in the C60 threads?

[AgeVivo]

Ok. Yesterday I fed the mice with C60oo
(I figured that if I do it it is essentially smoothing the transition from 1 dose per week to 1 dose every two weeks, but if I don't do it would create "what if AgeVivo had" questions here)

the mice are still doing well and still not as sportive as a few weeks ago. Thinking back (and as I had already written in this thread) they were really particularly exceptional when walking upside down. Now I clearly do not see that anymore. Walking up the platform as showed in the video of last week is really not frequent. I can't say what happens precisely because mice are not as expressive as we for example: when they hang on the platform and don't go up, it is as if they didn't want/there was no point to or perhaps as if it was too difficult anyway.

=> I think the mice are simply aging. Their hair is not as great as a few weeks ago. Their climbing capability is smaller (I clearly see it when they try to go on the top of the balls). It seems to me that their balance seems a little more fragile (to be confirmed with time, as it is tenuous: I see it when they stand on their legs on a slightly unstable ball). It seems to me they see slightly less well (when I put a piece of bread on the top of the cage they guess there is something but take time to find it; it might be that they are simply less "excited" or rotate their body less). All this is tenuous and gradual but when I look at my first threads and various images I think that after an impressive progress (it seems to me at least) their ability seems to me now a little below what it was before the experiment. Between last week and this week it is difficult to say if things have changed up or down (I would say down mostly, but not obvious). It is more the combination of both that make me say this.

After all at this age, in a large lab study a visible portion of mice would have in principle already died (http://agevivo.com/l...DGA-aspirin.gif). So in a very unscientific subjective guess my guestimate is that C60oo did/does something good to them but it doesn't stop aging. Some factors can not be excluded: the kitchen is slightly warmer now that it is the summer (by not much; 2 degres perhaps; plus they are not in front of the sun rays), to me it is perfect now but they could well be better with a lower temperature.

As always, here are pictures (didn't have a good camera with me today; the last picture is the best quality I could get (while changing water): we can see the hair)
 bb110812.jpg   23KB   6 downloads  w110812.jpg   28.93KB   6 downloads  bw110812.jpg   23.42KB   10 downloads  all110812.jpg   40.86KB   12 downloads

Edited by AgeVivo, 12 August 2012 - 11:59 AM.

[niner]

Today I came back from 2 week vacations. Good news the mice are doing well (of course this was expected; it is not the first time that I go on a vacations). Here is a video: http://agevivo.com/l...040812-mice.avi

Although I notice that they are less "sportive". Example with a video: http://agevivo.com/l...40812-sport.avi . Perhaps it is due to aging (scarcopenia, rhumatisms... aging), perhaps it is due to the lack of c60 for 2 weeks (coming back to a more normal state for their age?), perhaps it is due to the fact that I just came back and somehow disrupted their rest/cycle (during vacations I put a 12h day/12h night light in the kitchen); I'll try to keep an eye on their motricity in the coming days.

I am wondering if I should give them C60 next week-end or the week-end after. With the plan I scheduled planned it should be the later. But in case the reduced activity is confirmed during the week, I'd like to have a feeling of whether it is due to spaced c60 feedings.

Was your 2 week vacation between scheduled feedings, or did they miss a feeding? Do you know if they are getting the same dose in mg/kg as Baati's rats? I think the allometric scaling factor should have mice getting twice as much as rats, if I remember correctly.

[AgeVivo]

Thank you for your help niner.

[Was your 2 week vacation a) between scheduled feedings, b) or did they miss a feeding?

To answer exactly: with reference to Baati et al, we are talking about the transition from one dose every week to one dose every two weeks, so a) or b) depending on where the transition should exactly be (I started at age 18 months in mice and they started at age 10 months in rats: it is difficult to say where the transition should exactly be)

To answer what I believe you have in mind: indeed so far they had c60 every day then every 7 days... and for the first time they had to wait for 14 days.The Baati et al paper does not say if then found something simialr when they started feeding the rats every 14 days, but they started at younger ages so I guess they could not see effects of aging clearly.

Here I put a 1 when they receive a dose during a week-end and a zero otherwise:...1111011010101... The post you are refering too is the bold 1. In italics is what I have in mind for the future. I guess that if I want to learn I need to be [present and] attentive during the coming days. Today it seems to me that over the day they got a little more active, but if could be purely normal and they are still far from climbing up the hanging platform frequently.

Do you know if they are getting the same dose in mg/kg as Baati's rats? I think the allometric scaling factor should have mice getting twice as much as rats, if I remember correctly.

The researchers thought it would not be important (sometimes they inject massive doses). We had made a short calculation from which I had decided to give two drops to give a little more to mice than rats in terms of scaling. A few weeks ago I have weighted drops -- I need to find my notes and I am coming back in a next post with a quantitative answer...

Edited by AgeVivo, 12 August 2012 - 08:01 PM.

[AgeVivo]

Dose calculation
Ok, so I have weighted drops on a precision balance: 6 drops weighted 183 mg (=0.183 g).
The concentration of C60oo is 0.8 mg/ml (= 0.8 mg/g) and Mice weigh about 30g (= 0.03 kg)
So 2 drops is about 2*(0.183/6)*0.8 / 0.03 = 1,62 mg/kg.
When I look at the paper (abstract of http://extremelongev...0-Fullerene.pdf ) I get 1.7 mg.

Oh I am using the same allometric scaling factor as Baati et al but in mice! When we had done a short calculation I thought I was giving more. Hmm... We thought that drops would be much larger; I did things without the required time to set up everything. Pff... Should I increase the dose now, maintain it? I have to think but opinions are welcome.

What should it be?
Let's first imagine that we would like to do the correct experiment from scratch.
- Niner, do you remember niner where the times 2 factor that you have in your mind.
- Recently I have read (on GRG) that scaling should be done by body surface (q/s = Q/S) rather than by body weight (q/w = q/W). In terms of weight, between rats of w = 600 g and mice of W = 30 g so a factor of 20. the surface rule becomes (q/w)*r = (Q/W) * R so (q/w) = (Q/W) * (R/r) or (q/w) = (Q/W) * (W/w)^0.33. This leads that with the body surface rule I should use 1.7*20^0.33 = 4.6 mg/kg

In both cases quite more than what I am doing. That could well explain.

Edited by AgeVivo, 12 August 2012 - 09:24 PM.

[AgeVivo]

I have found an online tool http://web.ncifcrf.g...eConversion.pdf
=> if I had better prepared my experiment I should be using about 3.5 mg/kg so 4 drops instead of 2. (indeed a factor of 2 approximately).
That could well explain.

Edited by AgeVivo, 12 August 2012 - 09:53 PM.

[AgeVivo]

I could be wrong -- and I will doublecheck with the authors -- but I seemed to me that Baati et al had not chosen one particular dose: they could typically have chosen twice as less or twice as much. Therefore I am surprised that the dose would be the reason for what I see -- but it could. We will soon start mprize at home with c60 (pet owners have answered positively; if you are a pet owner or if you are interested in having mice or rats at home, PM me), and some will do the experiment with mice.

So I would like to use my experiment as a hint for a more robust mprize at home: I think that next week I will double the dose. I really don't like to change an experiment once it is started so I am still hesitating => if you have suggestions you are welcome.

[Logic]

I am still of the opinion that one should try to improve on the results obtained by Baati et-al who slowed and discontinued dosage to avoid having the experiment take too long IMHO.
It seems they may even have killed the last two rats to end the experiment.

Someone here mentioned the positive effects having worn off after 6 days.
So IMHO dosing every 3 days is required and one has to take mouse metabolism into account.

Why try to repeat their results rather than improve on them?

[niner]

I have found an online tool http://web.ncifcrf.g...eConversion.pdf
=> if I had better prepared my experiment I should be using about 3.5 mg/kg so 4 drops instead of 2. (indeed a factor of 2 approximately).
That could well explain.

Maybe. If we consider the amount that appears to be needed for humans to see effects from it (0.02 mg/kg daily in several of our people, roughly equivalent to 0.12mg/kg in rats or 0,24mg/kg in mice), the mice might be running low. Lister saw effects from 0.02mg/kg-d fade in about 6 days, and that was after daily dosing. The mice were already on a weekly schedule. If they perk up after their next dose, this might be the explanation. Or... It might just be due to the fact that the mice started a lot later in life than the rats did, and they're getting older. Baati's rats, although they lived a long time, may not have shown a lot of later-life vitality. There really wasn't any particular testing of that, so we don't know much about how the rats behaved later in life.

Someone here mentioned the positive effects having worn off after 6 days.
So IMHO dosing every 3 days is required and one has to take mouse metabolism into account.

Why try to repeat their results rather than improve on them?

Yes, I think there's something to that, but we have to take the dose into account. The more you take, the longer it should take to wear off.

At first, my thought was "but of course you want to replicate!". However, giving it some thought, we don't have to duplicate every single aspect of the experiment. AgeVivo isn't doing that anyway; he's using mice instead of Wistar rats, and they started much later in life. They are also being dosed in a more variable way, in that they can share the bread, while Baati's rats got a tube down their throats. The thing that we really need to replicate is life extension in mammals from C60-oo, not every detail of the experiment. As such, I see no problem with continuing the dosing on a weekly or bi-weekly schedule for the rest of their lives, which I think would give us a better chance of seeing significant life extension.

[Logic]

Thx Niner. Although dosing 2 or 3 times a week may be better considering their faster metabolism?
I dont know if anything above a certain (saturation point) amount isnt simply eliminated?

[Mind]

I am still for replicating Baati...because it was such a small sample size. There is soooooo much research out there that is erroneous, cannot be replicated, or even fraudulent, that it boggles the mind. With our Mprize@home and the pet owners, we are going to at least start out with replicating Baati. Once we have a good number of rats/mice in the study (maybe 40), then, if we can find more rat owners, we could do an additional "improvement study" and dose the rats all the way through de-animation.

Edited by Mind, 13 August 2012 - 04:49 PM.

[niner]

I am still for replicating Baati...because it was such a small sample size.

Well, I would argue that because the effect was so huge, and so consistent through all six rats in the C60 arm, the sample size was large enough. Small sample sizes are a problem when the effects are small. Of course a larger sample size would be nice, but the effect was already far beyond the usual minimum for statistical significance. If we modified the protocol, but still saw a substantial life extension, that could give us new knowledge that a duplicate of Baati wouldn't give us. OTOH, if we saw nothing with the modified protocol, someone could argue that we "did it wrong". However, we're already "doing it wrong", because the very nature of our methodology (random pet owners instead of a centralized facility, and probably some method of feeding that is more ad-lib rather than injection) precludes a perfect duplication.

[AgeVivo]

1. answer to posts
Thank you all for the comments.

They are also being dosed in a more variable way, in that they can share the bread, while Baati's rats got a tube down their throats.

- Actually what I do is quite precise: they don't share the bread. I make 3 pieces of bread, each with 2 drops of C60oo. And one after the other, I put one mouse in the cage only with one piece of bread only, and I wait until it is completely finshed (quite fast as they have not eaten for a few hours) before I go to the next mouse and next piece of bread
- In a sense it is much better than gavage. Gavage causes a high stress in rats (and the person doing the gavage...). Most of the cases it is perfectly Ok but if once they decide to react vigourously and get hurt in the throat then it gets worse and worse (kill them in very exceptional cases). I have ask the authors images/videos etc and I have seen a video of the technician doing gavage: the person was obviously very good at that. But still it may happen. In the paper they wrote:

As rats are known to be sensitive to gavages, we decided to stop the treatment for all rats and to observe their behaviour and overall survival

The thing that we really need to replicate is life extension in mammals from C60-oo, not every detail of the experiment. As such, I see no problem with continuing the dosing on a weekly or bi-weekly schedule for the rest of their lives, which I think would give us a better chance of seeing significant life extension.

I think so too. The two relatively-minor-after-all issues I see are:
1. I make them fast overnight before feeding them, which obviously has an effect especially if I do it lifelong. I think it is approx Ok (no such great effect if I interpolate lifespan extension by late onset caloric restriction and late onset intermittent fasting/ I do it by memory it would be nice if someone has precise numbers in mind)
2. The researchers told me that too much oil during too long is not good for health. They told me about steatosis and lipidemia, in the paper I see:

As biodistribution studies after daily gavages showed that C60 accumulates in livers and spleens, in order to avoid the negative effects of prolonged olive oil administration such as obesity, excessive steatosis, liver lipid degeneration, and insulin resistance [45], we treated the rats daily only during 7 days and weekly during the first two months, then every two weeks until one control rat died.

I am not familiar. with that. But we take salads with oild and vinegar ever day, Jeanne Calment was taking a lot of olive oil, the olive oil treated animals lived long so I would tend to think that we have much room left before it globally has negative consequences on their health.


2. Sharing my thoughts, globally
Tonight they seem like yesterday night. I remember when I first read about C60: I was thinking that it was perhaps a great joke; then I met the authors asked many questions, asked friends who studied close to them during that time etc and was convinced that it was clearly to replicate. Then when treated the mice started doing better and better and better and even do great things but at the time I refused to accept too easily/quickly that it could be working so well (placebo effect, some kind of extasy for mice, the overnight fastings...). I thought I would have more time to accept it slowly. Then they became not so extraordinary so now I think:
1a) wow they really were great (I am happy to have taken the video: http://agevivo.com/l...-very-small.mp4)
1b) now they are still ok but very, *very* far from being able to walk upside down (i have put a piece of chocolate cake that they can only get that way: but they don't go get it; though if I throw a little chocolate, they really fight for it. Clearly the video is like another world
1c) has something happened when I was out in vacations? Really I don't see it could. I have done that many times, everything was normal when I left and came back. They had water food day-and-night the temperature was normal, they had made constructions with straw..
1d) could #1a be explained by some hormonal effect of c60 where they wanted to look great and try everything... until they got aches everywhere and didn't want to try again?
2a) I am surprised that it could be the low-dose effect. But at least to test it (/rule it out perhaps): I need to really increase the dose, perhaps even more than by 2: the greatest activity I have seen could come from late effects of the initial treatment day after after day during one week.
2b) Perhaps the best way is to actually to continue and the mice will live long despite being not so great. But I have a hard time imaginating that. And there will always but "if"s anyway based on my experiment only. Increasing the dose is probably the best to see a reaction.
3) I will ask the authors if they know if their rats where particularly active and then not. But I have been in labs before and I know that we don't spend the time looking at the rats and that cages are too small for rats to do crazy things like mice do in my cage.
4) I realize how important my preliminary experience is for the mprize at home experiment to start well. Early Mprize at home pets will likely be pictured here in september, to see them well before treatments, and by the end of september I will have had collected much information with this small experiment to make more educated choices.


3. What's next for now
Next week end I will give 4 drops.

Edited by AgeVivo, 13 August 2012 - 10:18 PM.

[niner]

They are also being dosed in a more variable way, in that they can share the bread, while Baati's rats got a tube down their throats.

- Actually what I do is quite precise: they don't share the bread. I make 3 pieces of bread, each with 2 drops of C60oo. And one after the other, I put one mouse in the cage only with one piece of bread only, and I wait until it is completely finshed (quite fast as they have not eaten for a few hours) before I go to the next mouse and next piece of bread
- In a sense it is much better than gavage. Gavage causes a high stress in rats (and the person doing the gavage...).

Oh, good, that sounds like an excellent feeding plan. I think I must have gotten the wrong idea from one of your early posts. I like this better than gavage, due to the stress as you mention.

1. I make them fast overnight before feeding them, which obviously has an effect especially if I do it lifelong. I think it is approx Ok (no such great effect if I interpolate lifespan extension by late onset caloric restriction and late onset intermittent fasting/ I do it by memory it would be nice if someone has precise numbers in mind)
2. The researchers told me that too much oil during too long is not good for health. They told me about steatosis and lipidemia, in the paper I see:

As biodistribution studies after daily gavages showed that C60 accumulates in livers and spleens, in order to avoid the negative effects of prolonged olive oil administration such as obesity, excessive steatosis, liver lipid degeneration, and insulin resistance [45], we treated the rats daily only during 7 days and weekly during the first two months, then every two weeks until one control rat died.

I am not familiar. with that. But we take salads with oild and vinegar ever day, Jeanne Calment was taking a lot of olive oil, the olive oil treated animals lived long so I would tend to think that we have much room left before it globally has negative consequences on their health.

There have been a number of rodent feeding studies where animals were given a significant fraction of their diet as fat. Mice develop the usual diseases on such diets. The thing to do would be to figure out what percentage of their total caloric intake is coming from olive oil. As long as it is a sufficiently small fraction of the total, they should be ok, but I don't know exactly what the number should be. My recollection is that 20% fat was too much for mice, but I should try to find the paper. Since they're only getting a few drops of oil once a week or less, it's probably not a problem, but I don't know exactly how much they eat in total.

1c) has something happened when I was out in vacations? Really I don't see it could. I have done that many times, everything was normal when I left and came back. They had water food day-and-night the temperature was normal, they had made constructions with straw..

Because their behavior changed so much while you were away, this seems like at least a good candidate for an explanation. How were they fed while you were gone? Did someone come in and look after them, or did you just leave a lot of food and water? If you've done this for two weeks in the past with no problems, that tends to rule out things like psychological trauma because their human was gone. They would have experienced it before and would be used to it. Could they have just gotten sick? Caught little mouse colds or something? It's too bad that we don't have a control group for them.

[PWAIN]

AgeVivo, first thank you for running this experiment, I am sure that I am not alone here in my appreciation of your efforts.

Since your mice are different colours and easy to differentiate and you are dosing them independently, how about giving 1 mouse 2 drops, 1 mouse 4 drops and the last one 6 drops? This would allow dose dependancy to be guaged to a smal degree.

Edited by PWAIN, 14 August 2012 - 05:59 AM.

[AgeVivo]

Thank you. The mice seem slightly more sportive today that in the previous days. Slightly.

Niner, you are wondering and wondering like me. Having wondered much, overall my feeling is that they it is simply that they are old and ageing. Indeed, apart from the dose perhaps, or the way the outcome of this experiment should be (non-naturally-running elderlies), I haven't found so far other explanations that seem probable to me:

Because their behavior changed so much while you were away, this seems like at least a good candidate for an explanation. How were they fed while you were gone? Did someone come in and look after them, or did you just leave a lot of food and water? If you've done this for two weeks in the past with no problems, that tends to rule out things like psychological trauma because their human was gone. They would have experienced it before and would be used to it. Could they have just gotten sick?

I don't think that my absence has provoked something. I know it roughtly corresponds I have been on vacations many times, with these mice and others, without deterioration. Feeding is simple: I put a lot of food. Same for water. (they have a big water bottle with some methylene-blue-based fish-cleaning drops so that it doesn't turn bad), I have given details earlier in this post and other threads too, simple and effective.

Sick: I don't think neither. At least not like a cold and really more like aging .
Absence of natural light during two weeks? Although Baati et al has their animals in a lab (no natural light, expect when fed C60 I think)

Edited by AgeVivo, 14 August 2012 - 07:39 PM.

[AgeVivo]

Since your mice are different colours and easy to differentiate and you are dosing them independently, how about giving 1 mouse 2 drops, 1 mouse 4 drops and the last one 6 drops? This would allow dose dependancy to be guaged to a smal degree.

Hi PWAIN. Good question. Anyone some thought about it?

I first thought 'no' without hesitation (what do you infer from n=1? if your animal has some disease is it due to the treatment? I am already at n=3 only) BUT... here in this case, with the mprize at home with C60 starting soon (so this current experiment should be used to pave the road and test things for mprize at home) I have some doubt.

[Danail Bulgaria]

AgeVivo, how long is the average lifespan of the mice, that You are doing Your experiment with?

[Hebbeh]

Same for water. (they have a big water bottle with some methylene-blue-based fish-cleaning drops so that it doesn't turn bad)

Will the methylene blue taint any final results?