Journal Club…Lab lession, re-inventing the wheel to find lesions

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Tracking the evolution of CNS remyelinating lesion in mice with neutral red dye Maryna Baydyuk, David S. Cha, Jingwen Hu, Reiji Yamazaki, Evan M. Miller, Victoria N. Smith, Katherine A. Kelly, and Jeffrey K. Huang PNAS

To develop therapeutic strategies that enhance repair processes during central nervous system (CNS) injury, the molecular and cellular changes occurring at the injury site must be determined. However, precisely isolating the injury site, such as lesions in animal models of demyelination, has been difficult. We developed a simple and powerful method to track and analyze changes that occur within the lesion microenvironment over time. We demonstrated that neutral red-labeled lesions could be selectively isolated and processed for detailed analyses of transcripts, proteins, cell populations, and metabolites. The results of our study can be used to identify molecular pathways that promote CNS repair and the development of therapies to modulate neuroinflammation or enhance regeneration in demyelinating disorders such as multiple sclerosis.

Animal models of central nervous system (CNS) demyelination, including toxin-induced focal demyelination and immune-mediated demyelination through experimental autoimmune encephalomyelitis (EAE), have provided valuable insights into the mechanisms of neuroinflammation and CNS remyelination. However, the ability to track changes in transcripts, proteins, and metabolites, as well as cellular populations during the evolution of a focal lesion, has remained challenging. Here, we developed a method to label CNS demyelinating lesions by the intraperitoneal injection of a vital dye, neutral red (NR), into mice before killing. We demonstrate that NR-labeled lesions can be easily identified on the intact spinal cord in both lysolecithin- and EAE-mediated demyelination models. Using fluorescence microscopy, we detected NR in activated macrophages/microglia and astrocytes, but not in oligodendrocytes present in lesions. Importantly, we successfully performed RT-qPCR, Western blot, flow cytometry, and mass spectrometry analysis of precisely dissected NR-labeled lesions at 5, 10, and 20 d postlesion (dpl) and found differential changes in transcripts, proteins, cell populations, and metabolites in lesions over the course of remyelination. Therefore, NR administration is a simple and powerful method to track and analyze the detailed molecular, cellular, and metabolic changes that occur within the lesion microenvironment over time following CNS injury. Furthermore, this method can be used to identify molecular and metabolic pathways that regulate neuroinflammation and remyelination and facilitate the development of therapies to promote repair in demyelinating disorders such as multiple sclerosis.

I was sent this one by DrM&M…I saw the title and thought “That’s really interesting” then looked at the results and thought “That’s dilusional”.

The title is so misleading. So for my online journal club for people interested in science…read on…….. if not don’t

We want treatments that promote myelin repair, but to get them we need methods that detect de and then remyelination and we need to know where to look.’

When you dissect an MS brain you can see lesions and sometimes you sometimes see them in EAE too.

Guinea pig spinal cord with EAE lesions

So in this episode of Pee-NAS we have a paper suggesting that they have a tool to spot demyelinating lesions without having to do any histology. You give the dye and that tells you where to look. If you are injecting toxins into the brain to cause demyelinating lesions, you know where to look.

There have been umpteen imaging techniques that have made this claim, but none have made its way into routine use…probably says alot.

Here we have one that will help people working on the beasties

It is claimed that it is a useful demyelinating tool as it can be be spotted without any complex histology. This is useful if you want enriched lesions but sadly this is where the utility as a demyelinating dye stops!!

The dye is a vital dye and is taken up by living cell, other vital dyes can be taken up by dead cells as they can’t keep them out. There must be blood brain barrier dysfunction for the dye to get in . Then the dye must enter the cell and this preferentially occurs in macrophages/activated microglial and a few astrocytes. There is an imaging ligand that can do this to monitor hot lesions in humans and this is called “TSPO”. This can be picked up by a scanner.

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Now the problem with the title. Anyone who works with mouse EAE knows that the lesions are neurodegenerative and that there is not loads of demyelination and certainly there is no remyelination before the lesions develop, indeed there is not going to be demyelination before the lesions develop. But here perfectly normal EAE animals (B) have lesions similar to those in a paralysed animal (C). This is not suprising as lesions form before overt clinical signs develop. Therefore it is clearly not a dye of remyelination. If you read the paper it does not read that way. Perhaps the referees made the authors change this but they forgot to change the title.

It is not a seaslug but a spinal cord with peripheral nerves attached. The red (A-C) are the lesional areas

This is probably detecting blood brain barrier dysfunction and perhaps immune infiltration, but not demyelinationor remyelination, which is going to occur as a consequence to the attack. So the equivlent is more like a gadoliniuum-enhancing lesion in MS, but this dye costs a few pence verses having to buy a scanner for a few million squid.

There is of course the stone age approach to neutral red…its called Evans Blue.

This is a fluorescent dye that binds to a protein in the blood, called albumin (found in egg white) and gets in the brain where the blood brain barrier is leaking.

However, there are myelin dyes which can in theory be used to detect myelin in humans. However before they can be used, the chemical has to undergo safety testing as you need to make sure the drug is safe for human use. However, there is an MS drug that accumulates in myelin.

MS drug in a rat after (A) 24h or (B) 1 Week…Note white area in brain and spinal cord

Maybe this can be used to monitor myelination in humans?

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