Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia, 2023, Sara Cavaria et al

Some background information from the introduction that sets up their study - could be worth checking the references out:

(that's not about neutrophils - but might worth reading about. Have we heard about mitochondrial transfer?)

(Neutrophils are a type of white blood cell, as are macrophages, monocytes and lymphocytes. They are part of the rapid response system to invading particles and microorganisms. When the body is under stress, reserves of neutrophils are released from the bone marrow.

Image from: Neutrophil: A Cell with Many Roles in Inflammation or Several Cell Types? 2018
That reference highlights the heterogeneity of neutrophils - clearly there's lots left to know about them.)

 

Persistent Hypersensitivity in a Mouse Model of Chronic Widespread Pain
So, a model of chronic widespread pain?
What this seems to be is two injections of an acute inflammatory stimulus into muscle, 4 days apart - thus creating the 'Primed Mice'

The 'Control Mice' only had one injection. 7 mice of each.
Both sets of mice pretty much recovered after the first injection, in terms of sensitivity to pressure (50% mechanical withdrawal threshold), but the second injection caused the primed mice (in red in the chart below) to have much lower mechanical withdrawal threshold for the duration of the experiment, out to Day 28.



I'm not sure how relevant this is to fibromyalgia... but, onwards.

 

In vivo electrophysiology

The mice were anaesthetised,and the spinal cord exposed. Then the researchers applied stimuli (pressure and heat/cold) to the hind paw and measured the evoked activity of neurons. These measurements were done by an experimenter blind to which sort of mouse they were working on. I'm not sure when these measurements were done.

The researchers found that the neurons of the primed mice fired more than the neurons of the control mice when stimulated mechanically (prodded), but not when stimulated thermally. That does seem to be true, particularly for the measurements done with von Frey hair stimulus - which seems to be basically thin plastic rods of various stiffness which are poked at the mouse.

So, I think so far this only proves that inflamed tissue is more tender, which equates to the neurons firing at lower pressure thresholds.

 

Circulating Neutrophils Are Essential for the Development of Hypersensitivity in a Mouse Model of Chronic Widespread Pain
So, now the researchers wanted to find out what is causing this sensitivity of the nerves. This works was done in 4 control and 4 primed mice.

They transferred blood cells only and serum only from the control and primed mice to new mice. The top chart is for the blood cells, the bottom chart is for the serum - again it's the sensitivity to pressure analysis (50% mechanical withdrawal threshold). The p values are for the area under the curve for the full time course - only the blood cells from the primed mice caused a reduction in pressure thresholds in the new mice. There does seem to be good separation in the pressure thresholds for up to one day after injection into the new mice.

That's a new word for me - "algogenic" = "inducing pain".

At this point, the researchers don't know which blood cells are causing the pressure sensitivity. They sorted the cells into T cells, B cells, neutrophils and monocytes for both the primed and control mice, and repeated the experiment of transferring them into the new mice.

That looks to be true, (although the T cells from the primed mice also look to be having an effect when transferred, although it seems that that difference wasn't statistically significant).

Then the researchers removed the mice's neutrophils (using an antineutrophil antibody) prior to the day 4 second carrageen injection that created the primed mice. The neutrophil depletion lasted at least 24 hours; the researchers say that the application of the antibodies didn't affect pain behaviour. The mice treated in this way didn't subsequently show a lowered pressure threshold. The researchers also found that administering the antineutrophil antibody to the primed mice at day 28 reduced the hypersensitivity to the pressure for at least a day. Figure 3B - it looks pretty convincing.

 

I'm not surprised. I can't recall any reliable reports of elevated cytokines in ME/CFS or fibromyalgia, although perhaps there are some.

I'm not sure, has it been known before that neutrophils are associated with peripheral pain? And that removing them seems to prevent the pain? That bit seemed interesting and as convincing as samples of 8 mice in total could be.

 

Neutrophils Derived from Mice with Chronic Widespread Pain Infiltrate Sensory Ganglia

Somata? Plural of soma - the body of the neuron (and a few other cell types).


So, they found neutrophils had infiltrated the somata of the dorsal root ganglion in the mice that had had two injections of the inflammatory substance, whereas they had not in the controls that had only had one injection. The somata are a relatively long way away from the site of the induced inflammation.


Figure 4 ai.
It looks as though some neutrophils were in the dorsal root ganglion somata of the control mice (one injection) and the new mice (no injections), but there were a lot more in the primed mice (two injections). They say they also used FACS (fluorescence activated cell sorting) to confirm that.

They looked at cell surface markers - they found the neutrophils in the blood and in the neurons looked different in the primed versus those in the control animals. That's not surprising as the primed animals presumably have inflammation and the control animals don't. They also found that the neutrophils in the primed animals looked different in the blood compared to those in the neurons - that perhaps means something.

So, yeah, still waiting to see how this might be relevant to fibromyalgia.

 

Neutrophils Derived from Patients with Fibromyalgia Syndrome Induce Mechanical Hypersensitivity in Mice

The researchers then took neutrophils from people diagnosed with fibromyalgia (9) and healthy controls (5), and injected them into new healthy mice (4 mice for each person).

So, this is the key chart, I think, Fig 5c. The measure again is the mechanical withdrawal threshold of the injected mice when prodded with the rods of varying stiffness.



After an hour, the thresholds in the healthy mice given the fibromyalgia neutrophils were a lot lower than in the healthy mice given the neutrophils from healthy people. A small number of samples, sure, but that looks interesting, doesn't it?

 

For sure, it's a bad sign when the introduction makes claims that aren't supported by the references given for them. But, I think I said just the other day, it seems to happen in most papers.

It's the last section that I find most interesting. The healthy mice were treated identically, except that the human neutrophils were either from people with fibromyalgia or people without fibromyalgia. The pain response didn't change from baseline in the mice with neutrophils from healthy people, but it did change in the mice with neutrophils from people diagnosed with fibromyalgia.

I haven't had time to think about what might be wrong with this, other than bias in the researchers who were prodding the mice.

 

Ha. I wondered whether human neutrophils would work in mice, it seemed a bit amazing. I haven't read the discussion of this paper yet (or much of the Method - which is at the end of the paper. Surely, they will address that issue, it's pretty fundamental.

Not to minimise the impact of these studies on the poor mice, but this set of results seems to suggest more nuanced impacts than just across the board dreadful inflammation.

For example Figure 3: Baseline pain threshold is about 1 to 1.2. There might be some habituation over time, as by day 28 the thresholds were typically a bit higher.

The controls in Fig 3A just had the one injection of carageen, and seemed to bounce back from it ok, with their pain thresholds at least as high as at baseline, whether or not they had their neutrophils depleted at day 3 (clone versus PBS saline)
The primed animals in Fig 3B had two injections of caragreen (black arrows). The pain threshold of the ones that had their neutrophils depleted at day 3 bounced back up. But the animals that didn't have their neutrophils depleted clearly were very tender throughout.

(The Ipsi and Contra are just different locations where the pressure was applied.)