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#apaperaday: Chronic N-acetyl cysteine treatment does not improve respiratory system performance in the mdx mouse model of Duchenne muscular dystrophy

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Chronic N-acetyl cysteine treatment does not improve respiratory system performance in the mdx mouse model of Duchenne muscular dystrophy

A pick by Maxwell et al from Experimental Physiology on the use of antioxidant N-acetyl cysteine (NAC) in mdx mice. The study claims not to improve function…lets see about that. DOI: 10.1113/EP091862

Authors introduce that lack of dystrophin leads to chronic muscle damage, inflammation and fibrosis and oxidative stress in humans (Duchenne) but also in mice (mdx). This leads to impaired respiratory function – especially in mdx especially the diaphragm has pathology.

As oxidative stress is one of the key processes that maintains pathology, antioxidants are proposed as a potential treatment for Duchenne. NAC is an antioxidant that in the past has been tested in mdx mice. Short term treatment resulted in less inflammation and fibrosis.

It improved function & reduced muscle necrosis. NAC is safe in humans. In the past only short term (up to 6 weeks) treatment of 1-2% NAC in drinking water was tested. Here authors wanted to test more chronic use: 3 months testing in 30 (!) mice per group receiving 1% NAC in water.

Authors used large groups with and without NAC and they used only male mice, starting at 4 weeks and treating for 3 months. However, they did NOT use a wild type reference group…which makes drawing conclusions challenging. The treatment of NAC did not impact growth or body mass.

Diaphragm function was not improved by NAC treatment. HOWEVER, as there were no wild type reference what we do not know is whether the mice actually had diaphragm dysfunction at this age. If there is no deficit with wild type, there is no treatment effect expected.

So the fact that authors here do not see a treatment effect can mean 3 things: the treatment does not work, there is no deficit with wild type so function is not expected to improve, or both. Authors conclude the treatment does not work.

They also perform muscle EMGs in a separate group where they stress the mice by exposure to hypoxia and blocking nerves to part of the respiratory muscles. This did not show differences between NAC treated and control mdx mice. No wild types were included.

Here authors argue that it is known mdx cope less well with these stressed situations and they use historical wild type data. I would argue it is best to always include wild types in the same study as there are many parameters that can influence outcomes.

Furthermore, the fact that treated mice here do not improve also does not necessarily mean the treatment does not work, as these analyses are not per se physiologically relevant. Mice are put in superstressed conditions and these extremes may be too much to handle.

However, if these conditions never occur in real life, the translatability is very limited. Finally authors looked at histology: they see no difference in collagen deposition, infiltration and fibrosis between treated and untreated mice.

Several comments about the histological analysis: again a wild type reference would have been ideal. You would expect more pathology in mdx mice than authors see with the collagen deposition and H&E. There is a lot of collagen, but this could be the endo and perimysia.

The diaphragm has a relative high amount of connective tissue. I am used to seeing more affected diaphragms than this (which is why a wild type reference would have been good). Also for the H&E authors calculated %areas based on 4 regions captured by the microscope.

They do not mention how regions were selected and assessed (ideally by a blinded operator and selecting randomly rather than visually and ideally 2 independent operators). On RNA level they see a reduction of SOD1 (oxidative stress marker), suggesting NAC had some effect.

Authors discuss that this study shows long term treatment with NAC does not lead to an improved function and reduced pathology in the diaphragm of mdx mice. Normally I am all in favor of papers showing something does not work.

However in this case I do not know whether the conclusion follows the results. Lacking the wild type reference makes interpretation of the results and treatment impact very difficult. So for now, I remain undecided on whether NAC is therapeutic or not.

Authors do correctly point out in Duchenne patients NAC would be given on top of steroids (IF it ever reaches the clinic) – this is something that should/could be evaluated in the future authors suggest. Agree – but then I hope that authors will include also a wild type group.