Ageing is associated with increased vulnerability to environmental cold exposure. Previously, we identified the role of the cold-sensitive transient receptor potential (TRP) A1, M8 receptors as vascular cold sensors in mouse skin. We hypothesised that this dynamic cold-sensor system may become dysfunctional in ageing. We show that behavioural and vascular responses to skin local environmental cooling are impaired with even moderate ageing, with reduced TRPM8 gene/protein expression especially. Pharmacological blockade of the residual TRPA1/TRPM8 component substantially diminished the response in aged, compared with young mice. This implies the reliance of the already reduced cold-induced vascular response in ageing mice on remaining TRP receptor activity. Moreover, sympathetic-induced vasoconstriction was reduced with downregulation of the α2c adrenoceptor expression in ageing. The cold-induced vascular response is important for sensing cold and retaining body heat and health. These findings reveal that cold sensors, essential for this neurovascular pathway, decline as ageing onsets.

38 Ageing is associated with increased vulnerability to environmental cold exposure. Previously, 39 we identified the role of the cold-sensitive transient receptor potential (TRP) A1, M8 receptors 40 as vascular cold sensors in mouse skin. We hypothesised that this dynamic cold-sensor 41 system may become dysfunctional in ageing. We show that behavioural and vascular 42 responses to skin local environmental cooling are impaired with even moderate ageing, with 43 reduced TRPM8 gene/protein expression especially. Pharmacological blockade of the 44 residual TRPA1/TRPM8 component substantially diminished the response in aged, compared 45 with young mice. This implies the reliance of the already reduced cold-induced vascular 46 response in ageing mice on remaining TRP receptor activity. Moreover, sympathetic-induced 47 vasoconstriction was reduced with downregulation of the α2c adrenoceptor expression in 48 ageing. The cold-induced vascular response is important for sensing cold and retaining body 49 heat and health. These findings reveal that cold sensors, essential for this neurovascular 50 pathway, decline as ageing onsets. 51 52 Introduction 53 Upon exposure to cold, depending on the type and intensity, several counterbalancing 54 responses are produced, such as shivering thermogenesis involving skeletal muscle, or non55 shivering thermogenesis in brown adipose tissue (BAT) and peripheral vasoconstriction in skin 56 (Señarís et al., 2018, Morrison, Shaun F., Nakamura, 2011, Morrison, S. F., Nakamura, 2019). 57 To produce such responses, thermo-sensors in the form of temperature sensitive sensory 58 receptors are distributed throughout the skin and are considered to work as a first line of 59 defence against cold, which makes peripheral cutaneous responses a fundamental event in 60 the defence against environmental thermal challenge. The sensory receptors in the skin 61 initiate the vascular cold constrictor response which acts to protect against body heat loss and 62 prevent hypothermia. This response is followed by the subsequent vasodilation, a restorative 63 response that is essential to protect the affected skin against cold-induced conditions, such 64 as chilblains, trench foot, frostbite, and Raynaud’s condition (Daanen, van der Struijs, Norbert 65 R., 2005, Keatinge, 1957, Lewis, 1930). It is a finely tuned well balanced response that 66 maintains cellular function and physiological homeostasis during cold exposure. Whilst this 67 response is relevant to all ages, physiological changes in ageing leads to dysfunctional 68 signalling which causes a reduced adaptation to cold exposure (Guergova, Dufour, 2011). 69 With the lack of physical activity in the elderly population, it exacerbates the fall in core body 70 temperature which can cause fatal cardiovascular and respiratory problems (Billeter et al., 71 2014, Stares, Kosatsky, 2015). This is normally the biggest cause behind the National Health 72 Service (NHS) excess winter deaths that we witness every year, where in 2018 it caused 73 approximately 11,000 deaths linked to cold exposure in England (Office for National Statistics, 74 2019). 75 We have previously delineated the primary roles of transient receptor potential (TRP) channels 76 in producing a distinctive biphasic vascular response to cold in the mouse paw consisting of a 77 TRP ankyrin 1 (TRPA1)/ melastatin 8 (TRPM8)-initiated sympathetic α2c adrenoceptor 78 mediated neuronal vasoconstriction and a distinct TRPA1-CGRP (Calcitonin gene-related 79 peptide) mediated sensory-vasodilator component (Aubdool et al., 2016). TRPA1 is a 80 biomolecular sensor for reduced temperatures especially noxious cold (<18C), mediating 81 aversive behaviour such as avoiding cold-induced pain, whilst also being involved in mediating 82 inflammatory pain (Kwan et al., 2006, Nassini et al., 2014, Jain et al., 2011, Gouin et al., 2017). 83 Additionally, it activates C and Aδ sensory nerves to release neuropeptides such as CGRP to 84 mediate neurogenic vasodilation (Aubdool et al., 2016, Story et al., 2003, Gentry et al., 2010). 85 TRPM8 on the other hand is sensitive to low or reduced temperatures such as cool 86 temperatures (<28C) (McKemy, Neuhausser & Julius, 2002, Peier et al., 2002). It is involved 87 in deep body cooling and suggested to supersede the role of TRPA1 (Gavva et al., 2012). 88 TRPM8 is also suggested to be a vasoactive stimulus (Bautista et al., 2007, Johnson et al., 89 2005, Silva et al., 2019). The other established receptor that plays a pivotal role in cold 90 signalling is the sympathetic α2c adrenoceptor, which mediates the vasoconstriction of the 91 blood vessels (Bailey et al., 2004). Whilst the sympathetic branch that is involved in the 92 vasoconstrictor component of the cold response has been shown to have reduced activity in 93 ageing humans (Holowatz, Thompson-Torgerson & Kenney, 2010, Degroot, Kenney, 2007), 94 little is known about the functionality of the cold receptors TRPA1 and TRPM8 in ageing. In 95 the current study we hypothesize that signalling via the cold receptors TRPA1 and TRPM8 96 deteriorates with ageing which causes an impaired vascular response to the cold. 97 The primary objective of this study is to investigate the cutaneous vascular response to cold 98 in ageing, focusing on the activity of the cold TRP receptors; TRPA1 and TRPM8. As 99 sympathetic-sensory neuronal signalling is key for the cutaneous vascular cold response in 100 ageing, we also searched for evidence of dysfunction within these systems. Here using in vivo, 101 ex vivo, genetic, and pharmacological approaches we show that TRPA1 and TRPM8 signalling 102 declines with ageing which affects the sensing as well as functional pathways involved in cold 103 signalling; all of which contribute to the impaired cold vascular response. Additionally, we 104 provide evidence that the α2c adrenoceptor as well as the TRPM8 receptor both play critical 105 roles to influence this outcome, as the expression of both diminishes significantly in ageing 106 which impacts the vascular response to cold. These important findings establish the dynamic 107 role of cold sensitive TRP receptors and sympathetic receptors in the cutaneous vascular 108 response to the cold as ageing occurs. 109 110 Results 111 Cold-induced vascular response is impaired in ageing. We analysed the cold induced vascular 112 response in wild-type (WT) CD1 females (Young: 2-3 months, Aged: 13-15 months) with a full113 field laser perfusion imager (FLPI) using the cold water immersion model (Figure 1a) 114 developed in our laboratory (Aubdool et al., 2014, Pan et al., 2018). After the baseline blood 115 flow was measured for 5 min, the ipsilateral hindpaw was immersed in cold water at 4C, a 116 temperature that produces a robust vascular response, for 5 min and blood flow was then 117 recorded for another 30 min. The cold treatment produced a typical vascular response of rapid 118 vasoconstriction followed by a prolonged recovery vasodilator response in both young and 119 aged mice (Figure 1b-c, Figure 1-figure supplement 1a). In young mice, the cold treatment 120 produced a maximum vasoconstriction of 51.1 ± 1.1%, however, in aged mice this was 121 significantly blunted with maximum vasoconstriction of 27.7 ± 3.0% (Figure 1d). These 122 changes were reflected in the area under the response curve (AUC) analysis with a 123 significantly greater response in young than aged mice (Figure 1e). The result was extended 124 by measurement of the blood flow recovery after the cold treatment. Although blood flow did 125 not fully recover back to the baseline, the initial rate of recovery immediately after maximum 126 vasoconstriction before it slowly plateaued off was significantly faster in the young mice 127 compared to the aged mice (Figure 1f). These results suggest that with ageing the cold 128 induced vascular response starts to diminish, which affects both parts of the vascular 129 response. We were surprized that these changes were observed with moderately aged mice, 130 equivalent to middle aged in human terms (Dutta, Sengupta, 2016). However, at this age there 131 is a clear evidence of elevated gene expression in dorsal root ganglion (DRG) and skin of 132 senescence markers associated with ageing, P16; INK4A, cyclin-dependent kinase inhibitor 133 2A (P16) and P21; Waf1, cyclin-dependent kinase inhibitor 1 (P21), (Figure 1g-h) also 134 confirmed by western blotting (Fig 1i). 135 To extend our mechanistic understanding, we also used a laser Doppler imager (VMS-LDF), 136 in addition to FLPI, which simultaneously measures the blood flow, skin temperature and 137 tissue oxygen saturation level at a single point, to investigate the vascular response to cold. 138 Similar to the results obtained using the FLPI, the environmental cold water treatment 139 produced an impaired vascular response in the paws of aged mice compared to the young 140 mice (Figure 2a). In young mice, the cold treatment produced a maximum vasoconstriction of 141 45.5 ± 3.0%, however in aged mice this was significantly lower with a maximum 142 vasoconstriction of 23.4 ± 4.7%, a result which was reflected in the AUC analysis (Figure 2b143 c). There was a trend of greater reduction in skin temperature of aged mice after the cold144 water treatment; however, the aged mice had a significantly higher skin temperature at 145 baseline. This suggests that they were losing more body heat and is consistent with the fact 146 that the ability to maintain core body temperature declines with ageing (Figure 2d-f). The tissue 147 oxygen saturation level underwent a similar reduction in both young and aged mice after the 148 cold exposure (Figure 2g-h) but recovered more robustly in the young mice compared to the 149 aged mice as shown by AUC analysis (Fig 2i). We also found evidence of increased cellular 150 stress as protein expression of 3-nitrotyrosine (3-NT), a biomarker of oxidative stress 151 produced