Wednesday, 5 October 2016

Animal Experiments on film

Having been at a conference to look at science, it was inspiring and yet some aspects were rather depressing.

Why?...Because it was evident that we and others cannot compete with some labs because we do not have: their resources, sometimes their skill sets, importantly their ethical approvals (some of which I doubt we  would ever get :-( )  or their will to do such experiments.

As mentioned previously there is tendency to do EAE experiments three ways

Likewise there is often a prescriptive way of doing experiments (3 experiments of 5 a group...each probably woefully underpowered for each experiment and no sample size calculation) that likewise adds to lack of a 3Rs (reduction, replacement, refinement) approach (contrary to UK and now EU law on animal use)

If you want to see what people working on EAE are doing in terms of analysis. Have a look at the video by following the link. This type of analysis has become rather standard fayre, but adds to the costs and time taken to analyse the data.

Evonuk KS, Moseley CE, Doyle RE, Weaver CT, DeSilva TM.
Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination.
J Vis Exp. 2016;(115). doi: 10.3791/54348

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

In this study they aim to determine whether agents are immunomodulatory or neuroprotective and so I was excited to read how this is achieved. This is because any immunosuppressive agent will be neuroprotective because the immune response is inhibited and the immune response causes the damage and so no immune response means no TH1, Th17, demyelination nerve loss etc, but this does not mean that the drug is neuroprotective and protects damage to nerves despite the occurrence of the inflammatory attack. 

(a) They isolate cells from the CNS when they have disease and do staining of T cell markers Fox3P= T regulatory  IL17 = Th17 and gamma interferon = Th1 and look at T cell populations so it can see the good and the bad guys. However, if there is an immune suppression effect you get no or less cells and so every thing downstream disappears.  (They say this takes 2 days)

(b) They check proliferation in the periphery to see if the peripheral immune response is inhibited. If it is then you may get less immune infiltration. However you can get failure to affect proliferation and still get no immune infiltrate. Some antigens do not induce T cell prolieration in vitro.

If you look at the clinical score you can get this information as no signs invariably means no infiltrates and lots of signs means lots of infiltrates as they are directly related. 

The beauty of following of the signs is that you don't need to kill the animals to get an idea of what is going on (which is 3Rs) especially as it is changing from day to day and it changes in relation to whether the disease is waxing or waning. If you wait for the clinical signs to remit the cells have disappeared or are disappearing

(c) They then next look at the glial content and myelin content by analysing sections stained with glial markers or myelin stain to assess and demyelination (they say 2 hours per stain...However I doubt it as you have to cut the sections before you can stain and even if stain takes a few hours cutting sections from loads of mice will take some time).

However, this means that you are pre-selecting where things are happening. You could look at a lumbar (bottom of) cord but this does not tell you what is happening in the cervical (top of) spinal cord and a section at 10micrometer is only fraction of the 5 cm of spinal cord where the lesions can occur. So it would take 5000 sections to get an idea what is happening over all.

We use a system that looks at the whole cord and takes a couple of days to look at hundreds of samples which would take weeks if we did sections .....but often get told to look at a sections which is a 5000th of the information. 

Likewise using myelin stain in sections to equate demyelination (myelin loss with preservation of axon) misses the fact that white blood cells fill space and so create areas of loss of myelin but they contain no nerves. Likewise in many cases in the mouse there are no nerves present to myelinate because the inflammatory response has destroyed them.

But how do you work out what is immunosuppressive and what is neuroprotective? 

In the example here they suggest that to show the immunosuppressive effect you treat before the signs and to show neuroprotection then you treat at the maximum clinical disease after cells have infiltrated.

This is done by many EAEologists, notabaly in USA, however this experiment by numbers approach may not deliver.

In my humble opinion if you have a pure neuroprotective agent when treated before disease develops you will not get an effect on the degree of cell infiltration and the severity of maximal clinical disease will not be inhibited, however it will promote better clinical recovery because of inhibition of nerve loss. This may or may not be associated with a drop in myelin stain (at maximal disease the immune infiltrate will create areas of loss of myelin) and may or may not affect glial response as the main target is to look for nerve survival and in this case nerve content was not examined although it could be in (c) using a nerve stain. 

Treat at the maximum severity of disease and it may or may not give you benefit in the outcomes above although you would hope for more nerve content. However you cannot do the histology at the peak of disease and hope to get the answer about nerve protection, maybe you can do the same at the end of the experiment (but it means two groups of histology and perhaps another group to assess the clinical disease. We have seen that treatment after maximal disease is too late to give neuroprotection in some instances.  You would hope that the signs of disease would remit and stay below the vehicle group if neurodegeneration had occurred.

However, unfortunately I have to disagree that this approach is definitely going to dissociate an immunosuppressive from a true neuroprotective action. If we treat before disease and before the immune system nerve enters the CNS, there is no infiltrate no loss of myelin and no astrocyte response, no cytokines etc etc there is no egg so no chicken. However treat at peak disease and some immunosuppressive agents can still switch disease off within a few hours and so the neurological disease drops the cell numbers drop and the level of demylination and astrogliosis drops so the neuroprotection would be due to immunosuppression.
We can dissociate the two.

This current approach is the standard approach in MOG induced EAE in mouse. 

Therefore, every agent that is immunosuppressive is claimed to be a neuroprotective drug and drugs claimed to be neuroprotective may be immunosuppressive. Therefore, we over egg the pudding and drugs that inhibit the immune response work, where they would not in progressive MS.

A simple way to view this is to read the clinical disease, the peak indicates whethter the immune response has been blunted and the level of disability at remission indicates the amount of nerve loss. Remeber this when you look at EAE papers and you can't go wrong. However, in the current vidoe there is extra analysis which may lack 3Rs value. But it is what appears to be required in publications. 

Should we go with the flow or argue against this and save time, save money, save animals but struggle to publish as this is the way that EAE is done?

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