Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Induced by Repeated Forced Swimming in Mice. (Ohba 2019)

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Biol Pharm Bull. 2019;42(7):1140-1145. doi: 10.1248/bpb.b19-00009.
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Induced by Repeated Forced Swimming in Mice.
Ohba T1, Domoto S1, Tanaka M1, Nakamura S1, Shimazawa M1, Hara H1.
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1Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University.

Abstract
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by disabling fatigue of at least 6 months, in addition to symptoms such as muscle pain and muscle weakness. There is no treatment provides long-term benefits to most patients.

Recently, clinical research suggested the involvement of pyruvate dehydrogenase (PDH) in ME/CFS. PDH is a crucial enzyme in the mitochondria matrix that links glycolysis to the tricarboxylic acid cycle and oxidative phosphorylation. However, it is little known whether PDH could be a therapeutic target. The purpose of this study was to establish ME/CFS in mice and to investigate the involvement of PDH in ME/CFS. To induce the chronic fatigue in mice, a repeated forced swimming test was conducted. To evaluate fatigue, we measured immobility time in forced swimming test and starting time of grooming.

An open field test was conducted on day 8. After 25 d of the forced swimming test, the mitochondrial fraction in gastrocnemius muscle was isolated and PDH activity was measured. Moreover, we evaluated the effect of PDH activation by administering sodium dichloroacetate (DCA).

In ME/CFS mice group, the immobility time and starting time of grooming increased time-dependently. In addition, the moved distance was decreased in ME/CFS mice. PDH activity was decreased in the mitochondrial fraction of the gastrocnemius muscle of the forced swimming group. DCA treatment may be beneficial in preventing fatigue-like behavior in ME/CFS. These findings indicate that ME/CFS model was established in mice and that a decrease in mitochondrial PDH activity is involved with the symptom of ME/CFS.

KEYWORDS:
mitochondria; myalgic encephalomyelitis/chronic fatigue syndrome; pyruvate dehydrogenase; sodium dichloroacetate
PMID: 31257290 DOI: 10.1248/bpb.b19-00009

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Animals

All experiments were performed on adult male (7-weeks-old; Japan SLC Ltd., Hamamatsu, Japan) ICR mice. The temperature of the animal facilities was 24 ± 2°C, and the lighting was under a 12 h/12 h light/dark cycle (lights on at 8:00 h). Food and water were available ad libitum. All pro-cedures related to animal care and treatment conformed to the animal care guidelines issued by the Gifu Pharmaceutical University Animal Experiment Committee. The protocol was approved by the Committee on the Ethics of Animal Experi-ments of the Gifu Pharmaceutical University.Induction of ME/CFS Model with Forced

Swimming Te s t

The forced swimming test was conducted as previously described with some modifications.4,15,16) Each mouse (with tail loaded of 5% of body weight) was placed in a plastic cyl-inder (14-cm diameter) filled with water (3000 mL, 23–25°C) for 6 min for 25 d. Water was exchanged for each mouse. The movements of the mice were recorded by a video camera, and mice was judged to be immobile when they remained floating passively in the water, making only small movements to keep their heads above the water. The measurement of immobility time was performed in a blind manner by a single observer (SD). Control mice were not received any treatment.

Post-swim Fatigue

Post-swim fatigue was measured im-mediately after the forced swimming test. Each mouse was removed from the forced swimming apparatus and placed in clear observation cage. We measured the elapsed time until mice start to grooming (licking and rubbing of the skin/fur). The post-swim fatigue time was noted on days 1, 2, 3, 4, 5, 10, 15, 20 and 25.17 )

Open Field Test

The open field test was performed to assess locomotor activity at day 8 as described in detail with some modification.18,19) The test was performed using an open field apparatus (30 cm long ×30 cm wide ×30 cm high). Each mouse was placed into the apparatus and allowed to move freely for 1 h. The movements of the mice were recorded by a video camera. The total distance moved in the area was recorded using a computer-operated EthoVision XT system (Noldus, Wageningen, the Netherlands). The total distance moved in the area was used as index of locomotor activity.

Repeated Forced Swimming Did Not Affect Body Weight

We measured the body weight for 25 d after expo-sure to repeated forced swimming. The body weight of the mice exposed to the repeated forced swimming for 6 min/d for 25 d did not differ significantly from control mice (Fig. 1B). This result indicates that body weight change did not affect behavioral tests.

Repeated Forced Swimming Increased the Fatigue-Like Behavior in Mice

We investigated whether repeated forced swimming (load 5% of body weight, 6 min/d, 25 d) increased the fatigue-like behavior via behavioral tests. (Fig. 1A) We measured the immobility period during the forced swimming test to assess the fatigue-like behavior. An increase in the immobility period caused by repeated forced swimming was considered to be a behavior related to ME/CFS.20) The im-mobility period was significantly increased on days 5, 10, 15, 20 and 25 compared to day 1. (Fig. 2A) (degree of freedom; 8, F= 13.415) Next, we measured post-swim fatigue time im-mediately after the forced swimming test. The number of mice with post-swim fatigue time over 30 s was increased each day. (Fig. 2B) These results indicate that the repeated forced swim-ming induced ME/CFS-like behavior in mice.

Repeated Forced Swimming Decreased Locomotor Ac-tivity in Open Field Test

To investigate the effect of repeat-ed forced swimming on locomotor activity, we conducted an open field test at 8 d after exposure to repeated forced swim-ming. Representative images are provided in Fig. 3A. Total distance moved was lower in mice exposed to repeated forced swimming than in control mice (Fig. 3B). This result indicates that repeated forced swimming decreases locomotor activity.

Pyruvate Dehydrogenase Activity Was Impaired by Re-peated Forced Swimming

We investigated the involvement of PDH in this ME/CFS model mice. PDH activity was evalu-ated in the mitochondrial fraction of gastrocnemius muscle. The level of PDH activity of mice exposed to repeated forced swimming was significantly decreased compared to control mice (Fig. 4).

Efficacy of Sodium Dichloroacetate in Chronic Fatigue Syndrome

We examined the efficacy of DCA as an activa-tor of PDH in ME/CFS. We administered DCA (50 mg/kg, intraperitoneally (i.p.)) to mice during repeated forced swim-ming test (load 5% of body weight, 6 min/d, 15 d). The body weight of the DCA treated-mice did not differ significantly from saline treated-mice. (Fig. 5A) The immobility period of the DCA-treated mice was significantly decreased on days 5, 10 and 15 compared to the saline-treated mice. (Fig. 5B) DCA had no effect on immobility period on day 1. (Fig. 5C) These results indicate DCA suppresses the emergence of fatigue-like behavior. The level of PDH activity of DCA treated-mice was significantly increased compared to the ME/CFS group. (Fig. Screen Shot 2019-07-03 at 1.04.54 pm.png 6A). Maximum velocity of mice, which is used as an indicator of exercise capacity, was improved by DCA administration. (Fig. 6B) These results indicate that DCA might be beneficial for preventing fatigue-like behavior in this condition.


Screen Shot 2019-07-03 at 1.04.54 pm.png
 
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So this is pretty weird. Looks more like a treatment for overtraining syndrome than me/cfs. They should exhaust the mice, then give them a virus, then not exercise them for the rest of their lives and see if any of them continue to act exhausted anyway. That'd be a better me/cfs model.

Still, beggars can't be choosers and this experiment has *something* to it with the PDH analysis and DCA treatment.
 

ljimbo423

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So this is pretty weird. Looks more like a treatment for overtraining syndrome than me/cfs. They should exhaust the mice, then give them a virus, then not exercise them for the rest of their lives and see if any of them continue to act exhausted anyway. That'd be a better me/cfs model.

Still, beggars can't be choosers and this experiment has *something* to it with the PDH analysis and DCA treatment.
I would be really interested in the actual cause of the PDH impairment from the forced swim in these mice. I also agree that if their exhaustion lasted for the equivalent of at least 6 months to a year or longer, in human terms. That would be a much better model.
 

Wolfcub

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Interesting and valid findings here.
It just hurt my Soul to read it......

It figures. Why are many (though of course, not all) who are struck down with ME/CFS athletes in days gone by, fit and strong people. People maybe who pushed themselves....perhaps to breaking point?
I do know. I was always athletic, fit, strong, capable of walking many miles with no complaint or much tiredness, even into my 60s. Could run like the wind. Always did too much work. People "told me off for it" but I thrived on it...
Fought off viruses very well, hardly ever got sick. A cold only once in 3-5 years.

Then caught a virus, and the story goes way off track. Some tipping point was reached.
 
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Wishful

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To me it's another example of the lack of definition of ME. Is a decrease in PDH activity in muscle tissue proven to be a symptom of ME in 90+% of PWME? Is decreased PDH activity in muscles only caused by ME? I think they're overreaching their knowledge. The title should have been 'Decrease in PDH activity induced by ...', and then in the body of the paper they could speculate about whether it is the same mechanism as found in ME.
 
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To me it's another example of the lack of definition of ME. Is a decrease in PDH activity in muscle tissue proven to be a symptom of ME in 90+% of PWME?
The Japanese studies mostly focus on inducing fatigue via executive function (mental exhaustion) ; here they try forced swimming (yes, poor little animals) and they don't seem to recognize any viral contributation, or the Double Whammy theories (virus, then stress or mold or X factors)......

But the DCA aspect maybe worth further consideration...

In some of the other japanese studies I looked at they don't use the term ME or typical definitions.
 

godlovesatrier

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This really isn't useful. Why can't they focus on the biological exponents? Currently on twitter everyone is talking about mould and yet how many hundreds of thousands of people are exposed to mould and viruses and never develop ME? There will be a reason why our immune systems are prone to "breaking" - as WolfCub sort of sums up above, she was fine until the virus hit and she fought off colds easily. Whereas I did get colds regularly and did get sick regularly and was lethargic for 75% of my teenage years. Ironically the only thing that made me feel better after my teenage years was exercise. Exercise turned my life around at 16/17 to be honest, of course that ended when I got ME age 29. I really do believe that milder ME patients may hold the key. Mild ME patients are often invisible, doctors may not even have diagnosed them. But they dip in and out of remission constantly, there immune systems are broken one minute, ok the next. This may not last long, it may last for months. But why does it happen? I can't help but feel that this is an area of research that should be explored. For it might hold the key to understanding what the immune system is doing AFTER excercise is induced, to cause a sudden loss of immune function displayed as typical ME symptoms (flu like, stomach trouble, fatigue and eventually dizziness and ibs with organ slow down), where the patient may have felt almost normal (even if they were still none the less in the throws of ME) beforehand.
 

Wishful

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For it might hold the key to understanding what the immune system is doing AFTER excercise is induced, to cause a sudden loss of immune function displayed as typical ME symptoms (flu like, stomach trouble, fatigue and eventually dizziness and ibs with organ slow down),
I don't feel that there's a sudden loss of immune function during PEM. Exercise will increase IFN-g 24 hrs later, which can trigger other processes that cause the symptoms. The thread https://forums.phoenixrising.me/thr...chronic-fatigue-syndrome-komaroff-2019.76892/ hypothesises that the fatigue is due to cytokines affecting a specific part of the brain that controls the feeling of fatigue. Increase certain cytokines in that part of the brain -> feel fatigued. The 24 hr delay commonly experienced could be explained by the 24 hr delay before IFN-g rises. IFN-g triggers microglial cells into the active state, releasing more cytokines and chomokines, which then trigger this 'fatigue nucleus'. The other symptoms you list could also be due to downstream effects of IFN-g, such as an increase in neurotoxic kynurenines. So it's not a loss of immune function; it's just what is expected from (more or less) normal immune function. The response to (more or less) normal immune function is abnormal.
 

Shoshana

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Thank you for posting this for us to see, @Murph

Like others and yourself, I question if it has much if any, applicability to either humans, or to CFS/ME.

But I liked both of your follow-up qualifier posts. ;)

It seems to me, that even if we knew that a mouse result was equivalent to a human one, :woot:o_O
this research seems like it would be measuring the response to over-exercising, and recovery time from that, rather than it would have any resemblance or application to ME/CFS.

But as you noted, there is always a chance and we do not get all premium research for our illness, so far,
and @Wolfcub and other posters, had useful points, worth thinking about, as well.
 

godlovesatrier

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@Wishful Hi there. Thanks for your reply and your notes. All valid. But it doesn't explain why Dr Chia and Dr Lerner's studies proved successful at such a high rate. Warranted Dr Lerner had a higher rate of recovery in his patients whereas upon further inspection total recovery effecacy for Chia appears to be about 15 to 20% not quite the amount he touts in his literature.
 
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Mild ME patients are often invisible, doctors may not even have diagnosed them. But they dip in and out of remission constantly, there immune systems are broken one minute, ok the next
I agree with this. Davis is using the severely ill for his studies on the basis they will have 'a stronger signal'. Makes sense. But their strong signal will be on all the time. The charm of using mild patients is you can move them into and out of PEM and see what changes. And you can do it without major risk of breaking them for ever irreparably.

Given that we don't know what we're looking for and we're not sure what tests it will show up on, looking for something that correlates with PEM is likely to be the best way to find a signal and to trace that back to what is going wrong.

I'm extremely hopeful that it's in PEM that the core problem of this illness will be revealed, and I do feel a slight twinge of despair everytime a scientist studies blood taken on from a patient on a "good" day. We moderate our exercise to feel as normal as possible and that may be why the blood taken on those days is so rarely very different from controls.

Provocation studies are absolutely key. After all, it's only after provocation that Ron finds the impedance change.
 

Wishful

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But it doesn't explain why Dr Chia and Dr Lerner's studies proved successful at such a high rate.
Viral infections could add to the factors that create our symptoms. If our problem is something responding inappropriately to fairly normal levels of cytokines, then even minor viral activity could worsen symptoms. Getting a cold virus or viral meningitis greatly worsened my ME symptoms. I just wouldn't classify that as 'a sudden loss of immune function'; it's actually normal immune function. I'd classify my physically-induced PEM as an excessive reaction to normal immune response to muscle damage.
 

Wishful

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Provocation studies are absolutely key. After all, it's only after provocation that Ron finds the impedance change.
Some of us have unreliable responses, and some of us have such a high level of symptoms that it might be hard to see changes. Some of us have very reliable and abrupt changes in symptoms resulting from provocation. I certainly agree that the researchers should test some of this latter group. Analyze samples before exercise and again when I feel PEM peak. Then redo the tests with my PEM blocker. See what's different. I think they'd learn far more than by testing 1000 PWME at random times and trying to see what's different between them and healthy controls.

Add my vote for provocation studies.