Understanding pain mechanisms: RNAscope technology for RNA expression analysis

Learn how novel RNA in situ hybridization technology, RNAscope^®, can be applied to the analysis of human and rodent peripheral nervous system tissue to better understand the molecular mechanisms of pain.

In this SelectScience webinar, Prof. Theodore Price and Dr. Stephanie Shiers, of the Department of Neuroscience and Center for Advanced Pain Studies at University of Texas at Dallas, discuss technical aspects and challenges to using RNAscope^® technology for RNA expression analysis on rodent and human dorsal root ganglion (DRG).

Shiers describes her extensive experience working with human tissues from organ donors and how RNAscope can be used to gain molecular insight into pain mechanisms in humans. Price discusses recent advances in understanding how viruses might interact with the peripheral nervous system to cause pain or exacerbate viral disease.

This webinarfeatures:

• Human and mouse sensory neuron populations are distinct — which has important implications for pain mechanisms
• Type I interferons act directly on DRG neurons and increase their excitability
• RNAscope technology is a useful tool for analysis of human and rodent peripheral nervous system tissue

Read on for highlights from the live Q&A session or register to watch the webinar at any time that suits you.

Q: How do you deal with lipofuscin in human samples when quantifying signals?

SS: I touched on this briefly within the presentation. How the lipofuscin signal auto-fluoresces in the green, red, and far-red channels, means it is easy to rule out. This is because it appears white in an overlay image, or you can just toggle between channels and tell that it is not a real signal. However, I did not mention that we did try to block the lipofuscin with some known treatments, but you can use post-treatments. I have a lot of experience using Sudan Black, which is used to turn essentially the lipofuscin black. Lipofuscin is still there, but it is colored black and is undetectable in your fluorescence imaging. However, the problem with using that type of treatment is that it also turns your entire sample dark, which can reduce the fluorescence intensity of your real signal. So, when we are looking at tiny puncta or even puncta for mRNAs that are expressed at very low levels, it can be problematic. This could incur false negatives, as the intensity is reduced because of the Sudan Black or the TrueBlack post-treatment.

I have not used TrueBlack myself, although I have heard good things about it. This may be something we try in the future. But other than that, we mostly use lipofuscin to our advantage. This is because it resides in the neurons so it's easy to visualize the neurons that way, it's also easy to rule out because it does autofluoresce and alter colors.

Q: You show species differences between mice and humans. Are rats more like humans regarding the molecular features of the nociceptor subtypes?

TP: We published a paper when I was a graduate student on differences in CGRP and IB4 positive populations between mice and rats in the dorsal root ganglion and in the trigeminal ganglion. The short summary of that paper is that in mice, as Stephanie described, these two populations are largely segregated. In rats, they are not. But they are still not as overlapping as they are in humans. From this, it is fair to say the rat is somewhere in between. I think that performing a single-cell sequencing experiment would be the right thing to do on rats. Single-cell sequencing has been challenging in humans. I would be happy to talk about this further and explain why that is, if somebody has a question about that later. But I think a DRG single-cell sequencing experiment on rats would go a long way to help us understand at a global level how the populations of sensory neurons differ between mice and rats. Eventually, when we get to the point where we have that kind of high-throughput data for humans as well, then I think we will be able to do a lot using that information.

Q: Do you think that prolonged exposure to type I interferons may be a cause of neuropathy development after viral infections?

TP: Great question. Paulino Barragán-Iglesias is going to be working on that in his laboratory. He got a grant from the International Association for the Study of Pain to research exactly that. I am completely confident that Paulino is going to have some interesting results, from what I think will be a cool study. So, terrific question and look for Paulino to be publishing some great papers on that to come.

Q: When you talk about mouse expression, what mouse strain are you talking about?

SS: We use the C57 black 6 strain. We use males, and we use young mice. Like I pointed out in the Jeff Mogil paper with that figure. A lot of the mouse expression papers that are being published recently are using transgenic mice, that have a C57 black 6 background. We wanted to be consistent with that. We are also using younger animals. This means that these may not be the best representations of human DRG or the most homologous of tissue types or species types to choose. This is an inbred strain of mice. And we are looking at males only. Also, to note, we are looking at younger mice, which is like what other researchers in the field have used to study pain.

TP: I just want to add one thing to that. In this study, Stephanie did not do any direct comparisons with male and female mice. But we have done many other studies using female mice, looking at CGRP and IB4 colocalization and different neuronal populations. We have not seen any sex difference in terms of the neuronal populations, at least in the black 6 strain. I will also say that we have done similar studies in the past, again, not part of Stephanie's study here. But, where we have done CGRP and IB4 and outbred strains, etc., of mice, we have never seen considerable overlap between the two populations of mice. So, I think the segregation of the non-peptidergic and peptidergic in mice is likely to be true in males and females and in different strains. The age question is a great one. I have not seen anybody address that systematically, and we certainly have not.

Q: How do you quantify high and low levels of mRNA expression which can differentiate between different types of neurons?

SS: In the first paper we did not do a fluorescence intensity signal analysis. I just analyzed if it had a single punctum for a specific mRNA target, it was labeled as a positive neuron. So, that is true. It could be high expression or low expression or medium expression for the same RNA target, but we did not do that type of analysis. We are working out a way to try to measure abundance using area of the neuron that is covered with RNA or with punctate signal, to see if we can see any differences that way. However, that type of analysis is very time-consuming, and a lot more difficult to do than just labeling positives and negatives. This study is still ongoing right now and is something that I am working on.

Q: Are there differences between human subjects' age, sex, and medical history?

TP: We are trying to address that right now, mostly through this MD Anderson study. We had a paper that we published in ‘Brain’ last year that had a mix of electrophysiology and RNA sequencing on pain versus non-pain samples, from people having these vertebrectomy surgeries. At that time, we did not have enough samples to be able to say something about age or medical history, other than whether they had pain. But I would say that we are rapidly getting to that point. I cannot fully answer the question right now, but we are approaching it. We have close to approximately 60 human DRG samples that we have sequenced, where we have detailed medical histories and even some electrophysiological characteristics. I am excited about what we will be able to do in the relatively near future in that regard.

The other thing that is important is we have another bank sample for each one of those individuals. This means we can go back and do this kind of detailed cellular analysis on individual patients using the approaches that Stephanie talked about today.

Q: Do you know if a Nav1.9 channel participates in pain and inflammation pathways in SARS-CoV-2?

TP: I am pretty sure we do not even know whether SARS-CoV-2 is directly contributing to any kind of pain phenotype. There are some interesting studies out there. One that comes to mind is the paper published in Netarchive recently and will probably be published in other journals soon. This paper looked at the early symptom of loss of taste and smell. This symptom was found to be due to a loss of chemical sensation in their mouth. Many people get this symptom when they have been infected with the virus. This loss of chemical sensation is at least partially mediated by nociceptors from the trigeminal ganglion, which might also express ACE2.

However, no study on the scale of a full epidemiological study-type, shows that people with preexisting neuropathies get worse if they get COVID-19. We do not know at this point how long that will last. A lot of patients complain of relatively bad pain, but we do not have any idea what the mechanisms are, and if they involve Nav1.9 or not. We could look to see whether ACE2 is expressed in the population of neurons that also express Nav1.9, but we have not done that yet. I can tell you that they express Nav1.8 and Nav1.7 because it is a nociceptor population. And in humans, all the nociceptors or putative nociceptors express Nav1.8 and Nav1.7.

Q: Is N=3 used in human expression studies enough to address the variability between samples and patients?

TP: No, it is also hard to get enough of the tissue. I think eventually we'll be able to do this at a much larger scale, but for now, I can say — and Stephanie can comment too — one thing that has impressed me in the RNAscope work that we've done, is that there's remarkably little variation from organ donor to organ donor. So, most of the work that Stephanie talked about is DRGs that we have collected from organ donors, not from these patients from the MD Anderson study. I have been impressed that the variability in the normal populations is relatively small. Now, if you're talking about genes contributing to neuropathic pain phenotypes in the patients, I think that's going to be a lot harder to do with a relatively small N. Therefore, we have been building these data sets up as much as we can. Our goal, and our grant that just got funded by NIH, is to have 160 patients enrolled over five years. Given that we have already enrolled about 70, I am confident that we will be able to hit that number. And I think we will be able to learn a tremendous amount from what we do, but we are just scratching the surface on that project right now. We have a lot of work to do.

SS: I think the low variability is mostly dependent on the RNAscope assay and the quality of the mRNA in those samples. If you look at some of the classic NC2 papers with N sample sizes of say 10 to 12 human DRG samples, there is a lot of variability. I am guessing they had to use a large N because they were getting some bad quality tissue. With the RNAscope technology, as I went over with the positive control, we can gauge the quality of the mRNA. I think that has really helped us with getting these nice, low-variability data sets. I only showed an N3 for a lot of these different targets, but we now have probably six or seven human DRGs. And for all of these, with the histological markers we have been using, we are not seeing any differences. We also have a wide range of ages, races, sex, from patient to patient from these donors. The lack of variability could be gene-specific, we have not looked at them yet.

TP: I think it's also important to note that a lot of the papers that were published previously were using postmortem tissue which you would expect there would be a lot of variability in the RNA quality. Rob Gereau's lab and the folks at AnaBios completely changed the way that we do this by opening the idea of doing it from organ donors. Right after the organ donation surgery is over, you go in and take the DRGs and spinal cord, this allows you to have pristine mRNA quality in most of the samples that you obtain. I think this has been a game-changer. All credit there goes to those two groups, Rob Gereau's group at WashU and AnaBios.

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