The influence of TRPA1 signalling on the response to cold exposure as ageing occurs.

Recent studies in our group have revealed that the response of skin blood vessels to local noxious (10 degrees C) cold exposure is stimulated by a channel 'TRPA1'. TRPA1, when stimulated by cold, allows ions (e.g calcium) to enter and activate cells. The cooling of the lower limb of anaesthetised mice to 10 degrees C for 5 minutes leads to a decrease in skin blood flow, due to TRPA1-mediated constriction of skin blood vessels, to maintain heat in the body. Our studies have discovered the signalling and chemicals that are involved in this 'sympathetic' nerve-mediated pathway. Once the cold stimulus is removed, there is an increased blood flow response 'vasodilatation' to restore the blood flow to the skin and rewarm the affected area. This active recovery phase may help to prevent cold- induced injuries (e.g. frostbite). We have discovered that this rewarming phase is also due to an active role of TRPA1. This second phase involves TRPA1 stimulating dilator nerves, the 'sensory' nerves, to release small protein (peptide) transmitters espectially 'CGRP'.
We consider that TRPA1 has a major influence on the response of the body to environmental cold exposure. The peripheral effects in skin and the systemic effects in the whole body, in terms of core body temperature are directly linked. However, the role of TRPA1 in the link and as ageing progresses is unknown. We have pilot data that this response is largely lost in 13-16 month old mice (equivalent to late middle-aged humans).
The first sections of this project are designed to understand which components of the cold-induced vascular response in skin are lost as ageing progresses and how this impacts skin health. A series of experiments is planned, using anaesthetised mice to determine the activity of TRPA1 in ageing skin, whether it loses its ability to detect noxious cold as ageing progresses, or whether one of the phases of the vascular response no longer operates. In order to learn how the loss of the TRPA1-mediated vascular response to cold impacts on skin health, we will study skin blood flow, temperature, oxygenation and search for markers of tissue injury following local limb cold exposure, all in the same mouse paw. This will allow us to elucidate the biological mechanisms that occur and how these differ with ageing.
The loss of core body temperature, leading to hypothermia, can be fatal, especially among the elderly. We have developed a new technique to define the systemic response to whole body cooling on the unrestrained conscious mouse. We have exciting pilot data, using implanted radio-telemetry probes that sense body responses. When TRPA1 is lacking, the mouse is protected against the loss of core body temperature and onset of hypothermia. This suggests to us that although TRPA1 can kick-start vascular protection mechanisms in skin, it can also mediate accelerated cooling of the body. Whilst the role of the sympathetic nervous system in the whole body is established, much less known about the role of TRPA1 or the role of the TRPA1-CGRP pathway in whole body temperature regulation. We will examine how the TRPA1 is involved through using mice with TRPA1 genetically removed or TRPA1 blockade with experimental tools on the influence of ageing.
These studies will allow us to learn how TRPA1 influences mechanisms involved in cold-induced injuries in the periphery and whole body temperature regulation. The goal is that an improved understanding of these fundamental biological mechanisms will allow the chance to develop new approaches to control temperature regulation.

TRPA1 is a vascular cold sensor in skin, but pilot data shows that this response is deficient in moderately aged mice (13-16 months old). Pilot data has also revealed that TRPA1 deletion protects against core body temperature loss We will utilise murine models, to address how TRPA1 influences cold thermoregulation and what happens as ageing occurs.
We use non-invasive imaging to determine skin blood flow. In addition to genetically modified mice, we have available selective pharmacological ligands, and protocols for their effective use. We will expand techniques to measure local skin temperature, oxygen levels and changes to skin biology. Studies will be supported ex vivo with gene and protein expression, immunohistology and cell signalling experiments. All protocols are ethically approved internally and by the Home Office.
Firstly, we will compare nociceptive responses to TRPA1 in the young (2-3 month) and ageing (13-16 month) mice and learn of comparative localisation of TRPA1. We will investigate blood flow pathways down stream of TRPA1 cold activation, namely the noradrenergic sympathetic constrictor pathway and the sensory dilator CGRP pathway and elucidate the deficient mechanisms in the ageing cohort. To understand implications on skin health, we will measure blood flow, temperature changes and oxygenation concomitantly following cooling in the young and aged mouse paw, before ex vivo analysis for inflammatory cell and cytokine markers. We will investigate for a role for the alternate cold-sensing channel, TRPM8 and whether it may influence skin health, independently of effects on blood flow.

Finally we will utilise a climatic chamber and implantation of advanced radio-telemetry probes for whole body cardiovascular studies. We will measure parameters to learn how systemic cooling affects the ageing phenotype, before examining skin responses as rewarming occurs. Critically, we will examine the effect of a TRPA1 antagonist on whole body cooling.

(1) TRP researchers/scientists
There is a large community of TRP receptor scientists and the results will allow us to build on our recent results that TRPA1 is a vascular cold sensor. There is ongoing debate about the importance of the TRP receptors as cold sensors, as several have overlapping activities. The most immediate beneficiaries are scientists involved in the study of TRP channels and biological activities and their roles as biomolecular sensors. These scientists comprise a range from those studying fundamental mechanisms, to those involved in the translation of knowledge into drug development projects. Our positioning, in terms of expertise in applied murine models, means that we can tease out important mechanisms. From our previous experience in this area (CGRP, TRPA1, role of sensory nerves) these mechanisms translate well to human science.
The impact will be created via our publications and wider communications within the science areas. We will publish as soon as feasible in high impact journal and attend specialist as well as general science meetings, to ensure that the full potential and significance of our findings are realised. Discussions at these meetings will involve clinical and basic scientists involved in the more general treatment of the cutaneous circulation (either associated with excess cooling or injury) and with clinicians involved in treating systemic conditions associated with cooling, (hypothermia and the elderly).

(2) Industry
Most major pharma and some biotech companies have TRP discovery projects. These have been more difficult to deliver on than had been anticipated. The best adverse example of this, to date, is that the early generations of TRPV1 antagonists (developed like many of the TRP-related drugs for pain relief) were hyperthermic. The reasons were much debated. This has been costly for the companies involved and the programmes have had to wait whilst knowledge from basic science has caught up. Thus fundamental studies such as this are important to obtaining a full understanding and we continue to find interest in colleagues from industry when we present our findings. TRPA1 antagonists are now in clinical trials. There is also a potential to make monoclonal antibodies for TRP channels such as TRPA1. The progress to date has been slow, but an improved understanding of their adverse effects and further indications for their potential use would heighten interest in this area.
The present primary target for TRPA1 therapeutics is for the relief of pain, including that involved in diabetic neuropathy. Of note the main treatment at present for hypothermia is rewarming, which can be problematic. Thus the results from this project could influence drug discovery pathways. This would be achieved if it becomes clear that TRPA1 has pivotal roles in either the health associated with cooled skin or in enhancing the hypothermia, or the systemic response to the cold.

(3) Education, training and the general public
We are heavily involved in educating and training young scientists, starting by developing enthusiasm for the subject. TRP channels are of interest as spices activate them, including cinnamon and mustard oil for TRPA1. This is of interest to the general public, especially how this links with functional biology. Channels are also activated by a range of endogenous substances, in addition to noxious cold. This aids discussion with the public, especially the young, in addition to allowing trainees who visit the lab a general foundation on which to build the understanding of the science.

(4) 3Rs and refinement of animal models
We consider that our model of whole body cooling and analysis for mechanisms is more refined in terms of reduced stress and accuracy when compared with previous models where TRPA1 has been studied.