Hope for those who feel pain from touch

Elucidation of the mechanisms responsible for mechanical allodynia is

incredibly important in understanding and eventually treating the touch
evoked pain as the current opioid treatments are largely ineffective.

Neuropathic pain is a condition where damage

occurs to the nerves responsible for
information transfer between the brain and
spinal cord. A symptom of it is mechanical
allodynia, painful sensation from innocuous
stimulus. Things as simple as putting on a
cotton t-shirt can cause pain. Allodynia is
experienced by 15% to 50% of people with
neuropathic pain1. Common after cancer,
diabetes, chemotherapy, infection, or traumatic
injury.

In this 2021 study2 they identify a subset of

inhibitory interneurons in the Spinal Dorsal
Horn (SDH) that act as a critical brake on
conversion of A beta (touch) signals into pain-
like behaviour that is morphine resistant.
Providing a potential therapeutic target by way Figure 1: Circled in yellow shows the location of disinhibition
of activating these neurons may provide relief that occurs as a result of peripheral nerve injuries. It is the
inhibitory signals sent to lamina 1 neurons from Aβ fibres
to those suffering from the debilitating that allows the conversion of touch to pain.5
condition.

In a person without a peripheral nerve injury (PNI), high threshold afferent fibres are the ones
responsible for stimulating pain by transmitting to lamina 1 and nociceptive projection neurons and
then to the brainstem. However, in patients experiencing allodynia, this pathway gets highjacked by
primary afferent low threshold mechanoreceptors like Aβ fibres, considered the mediators of
mechanical allodynia. Previously the mechanism by which the signal from the Aβ fibres was
converted to pain was a mystery. 2Tashima et al.’s study reveals the subset of interneurons
responsible for the conversion.

No current explicit mode of pathology has been wholly determined, possibly due to difficulty in
manipulating Aβ fibre function in alive, awake animals. To get past these difficulties a transgenic rat
line, W-TChR2V4, was used. These rats have channelrhodopsin-2 (ChR2) (ion channels derived from
algae where they act as sensory photoreceptors) expressed at nerve endings of Aβ fibres associated
with specialised mechanoreceptors. Meaning that scientists were able to elicit a touch like response
by illuminating rats with blue light due to the ChR2 light gated ion channels. Using these new
optogenetics combined with cell type selective labelling, florescence, and electrophysiological
techniques Tashima and colleagues were able to characterise the neurons. Inhibitory interneurons
neurons located in lamina IIo with a tonic firing pattern and monosynaptic and polysynaptic
connections.

The neurons identified by the tailored adeno associated vector (AAV) were a subset of spinal dorsal
horn interneurons that receive information from Aβ fibres and inhibit lamina 1 neurons. Using
previous knowledge that Neuropeptide Y (Npy) in the spine has inhibitory effects on mechanical
hypersensitivity induced by a PNI3 the AAV construct incorporated a Npy promoter so that any genes
of interest chosen to add will be expressed in cells that express Npy. By microinjection of the vector
AAV-NpyP+ associated with tdTomato, red florescence helped identify the location and type of cells
with Npy.

A PNI was modelled in the rat participants to test what alteration to the pain pathway a PNI causes.
This model was developed by ligating and cutting the left L5 spinal nerve. After the PNI, whole cell
clamp studies were carried out, these exhibited a significantly lower resting membrane potential of
AAV-NpyP+ neurons and reduction in strength of input causing a decrease in excitation. It is
theorised that alterations in potassium ion flux after PNI is involved in reducing AAV-NpyP+ resting
membrane potential resulting in impairment of the Aβ inhibitory signalling to lamina 1 neurons.

All experimentation involving animals should be taken with a grain of salt, we cannot ask them if it
hurts. However, we can observe painlike behaviours like the paw withdrawal in this study. Inhibiting
the neurons with a toxin to test the effects on the behavioural response to four kinds of stimulus:
heat, cold, von Frey filaments and blue light. The withdrawal was given a score 0 to 2 to get more
precise results. This paw response was assumed to be due to a pain sensation however, self-
confessed in their discussion, this cannot be confirmed or denied. So, it was presumed along with
evidence of other aversive behaviour, that this showed a pain like response resembling mechanical
allodynia.

Induction of Aβ derived pain like behaviour was seen by inhibition of the SDH interneurons defined
by AAV-NpyP+. Adding a toxin receptor to the AAV construct and applying the toxin they were able
to inhibit the neurons in two different ways. After application of Diphtheria toxin (DTX) the rats
exhibited hypersensitivity to punctate mechanical stimuli that was morphine resistant. This was also
true for acute inhibition by clozapine N-oxide.

There was alleviation of the Aβ fibre derived neuropathic pain by activation of AAV-NpyP+ SDH
interneurons. Proven by adding a specialised ion channel to the AAV construct and applying a
designer drug, PSEM89S, that opens the channel activating the neurons. The paw withdrawal score
decreases, and the pain threshold increases, these are only momentary, and the effect wore off as
the drug does. C-FOS, a characteristic marker of depolarising neurons, showed that the number of
neurons firing significantly decreased in the superficial laminae of the ipsilateral SDH but not the
other laminae. Chemogenic activation of these neurons was able to alleviate the neuropathic pain
like behaviour experienced by the rats. Behaviour now known to be associated with a lack of the
signalling in the neurons defined by the AAV-NpyP+.

Another study investigating the same condition only using a different group of genes reported a
subset of excitatory SDH interneurons that regulate morphine resistant pain evoked by brush
stroking via excitatory superficial SDH signals4 Indicating that over the two reports a
counterbalancing of excitatory and inhibitory signals in lamina 1 neurons may be pivotal to touch-
pain conversion.

Additionally, wheat germ agglutinin, a transsynaptic anterograde tracer used as part of this study
revealed connectivity between the AAV-NpyP+ interneurons to deeper laminae where both noxious
and innocuous neurons receive stimuli then relay this to the brain. Pointing to the potential for
further discovery in the connections of deeper lamina neurons.

Finding a method of specifically activating the interneurons defined by the AAV-NpyP+ construct
could be the next therapeutic strategy for Aβ mediated neuropathic pain and other pain symptoms.
References
1. Prof Troels S Jensen, D. N. et al. Lancet Neurol., 13(9), 924-935, (2014).

2. Ryoichi Tashima, K. K. et al. Proc Natl Acad Sci U S A, 118(3), (2021).

3. Solway, B. B. et al. Proc Natl Acad Sci U S A, 108(17), 7224-7229, (2011).

4. Cheng L, D. B. et al. Nat. neurosci. 20(6), 804-814, (2017).

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